Antibodies to m(h)dm2/4 and their use in diagnosing and treating cancer

ABSTRACT

The present invention relates, inter alia, to certain anti-M(H)DM2/4 antibodies (including chimeric and humanized antibodies) or antigen-binding fragments thereof, pharmaceutical compositions comprising anti-M(H)DM2/4 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising anti-M(H)DM2/4 antibodies or antigen-binding fragments thereof bound to a cytotoxic drug, and the use of such antibodies, fragments, compositions and conjugates for treating cancer and/or for preventing metastases. For example, described herein are certain antibodies (including chimeric and humanized antibodies) or antigen-binding fragments thereof that specifically bind to extracellularly accessible epitopes of M(H)DM2/4 and inhibit tumor growth in vivo, pharmaceutical compositions comprising such antibodies or fragments, antibody-drug conjugates comprising such antibodies or fragments, and the use of such antibodies, fragments, compositions and conjugates for treating cancer or for preventing metastasis.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International Patent Application No. PCT/US2018/043908, filed Jul. 26, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/537,914, filed Jul. 27, 2017, which are incorporated herein by reference in their entireties.

2. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Sequence Listing filed with this application as an ASCII text file entitled “14160-007-228_SEQ_LISTING” created on Jan. 30, 2019 and having a size of 170,676 bytes.

3. FIELD

The present invention relates, inter alia, to certain anti-M(H)DM2/4 antibodies (including chimeric and humanized antibodies) or antigen-binding fragments thereof, pharmaceutical compositions comprising anti-M(H)DM2/4 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising anti-M(H)DM2/4 antibodies or antigen-binding fragments thereof bound to a cytotoxic drug, and the use of such antibodies, fragments, compositions and conjugates for treating cancer and/or for preventing metastases.

4. BACKGROUND

The MDM2 (MDM2 is a mouse homologue of HDM2) protein is composed of 489 amino acids and contains a p53 binding domain, two nuclear localization signals (amino acids 176-182 and 464-471), and zinc-finger motifs (amino acids 297-326 and 436-477) (see UniProt website, at UniProt Accession No. P23804). Its human homologue, HDM2, is composed of 491 amino acids and contains a p53 binding domain, two nuclear localization signals (amino acids 179-185 and 466-473) and zinc-finger motifs (amino acids 299-328 and 438-479) (see UniProt website, at UniProt Accession No. Q00987). Mouse protein MDM4 (also identified as MDMX) is a homologue of the MDM2 protein (see UniProt website, at UniProt Accession No. 035618), and both MDM2 and MDM4 are major negative regulators of p53 (Wade et al., 2013, Nat Rev. Cancer 13:83-96; Marine et al., 2004, Cell Cycle 3:900-904; Momand et al., 2011, Gene 486:23-30). HDM4 (also identified as HDMX) is a human homologue of MDM4 (see UniProt website, at UniProt Accession No. 015151). The most conserved domain within all M(H)DM2 and M(H)DM4 proteins is the RING domain which is responsible for ubiquitination of its target proteins, including p53 protein, and heterodimerization between M(H)DM2 and M(H)DM4. M(H)DM4 is required for M(H)DM2-mediated polyubiquitination of p53. A distinctive feature of M(H)DM2 and M(H)DM4 are their very complex expression pattern. Multiple-sized transcripts and protein products of M(H)DM2 have been identified in cancer cells by a number of groups (Olson et al., 1993, Oncogene 8:2353-2360; Bartel et al., 2002, Cancer Cell 2:9-15 (“Bartel 2002”); Sigalas et al., 1996, Nat. Med. 2:912-917 (“Sigalas 1996”); Iwakuma & Lozano, 2003, Mol. Cancer Res. 1:993-1000 (“Iwakuma & Lozano 2003”)). Many types of human cancers overexpress MDM2 protein and a common characteristic among these cancers is an associated increase in mdm2 splice variants. These M(H)DM2 variants have been shown to be expressed in a variety of tumors such as human ovarian, bladder, breast and astrocytic neoplasms, glioblastomas, leukemia and pediatric Rhabdomyosarcoma tumors (reviewed by Iwakuma & Lozano 2003; Rosso et al., 2014, Subcell Biochem. 85:247-61 (“Rosso 2014”)). Most interestingly they have been found to be more frequent in tumors of advanced stage (Bartel 2002). The multiple-sized M(H)DM2 transcripts that have been shown to be splice variants forms of the M(H)DM2 mRNA have been reported to be expressed more frequently in tumor cells than in normal cells (Bartel et al., 2004, Mol. Cancer Res. 2:29 (“Bartel 2004”)). It has been proposed that a mRNA surveillance system exists in untransformed cells, which degrades spliced transcripts and protects the cells from errors of transcription, mRNA processing, or mRNA transport whereas in transformed cells this system may not be functioning correctly (Bartel 2004). Moreover, some of these variants in cancer cells encode protein products, which have been shown to transform NIH3T3 cells in vitro, and some promoted tumor formation in mouse model (Sigalas 1996; Volk el. al., 2009, Mol Cancer Res. 7(6): 863-869).

To date more than 70 different M(H)DM2 splice variants have been identified (Bartel 2002; Bartel 2004; Rosso 2014). Some of the variants, for example MDM2-A and MDM2-B, are common to several tumor types (Sigalas 1996). Others have only been found in specific tumors, for example MDM2-FB25 and MDM2-FB26 in pediatric rhabdomyosarcoma. Several short forms of these alternatively spliced MDM2 transcripts correlate with high-grade malignancy in human ovarian tumors, bladder carcinomas and astrocytic tumors (Sigalas 1996; Matsumoto et al., 1998, Cancer Res. 58:609-613; Tamborini et al., 2001, Int. J. Cancer 92:790-796; Steinman et al., 2004, JBC 279:4877-4886). It has also been shown that aberrant mdm2 (281-, 254-, and 219-bp) and alternative mdm2 (653-bp) splice products strongly associated with shorter overall patient survival in breast cancer (Lukas et al., 2001, Cancer Res. 61:3212).

Several studies have evaluated the cellular localization of various human and murine M(H)DM2 protein variants to predict potential activities. In 25% of non-small cell lung carcinomas, M(H)DM2 variants were aberrantly localized to the cytoplasm (Evans et al., 2001, Oncogene 20:4041-4049). This result led to the discovery that the cytoplasmic compartmentalization of the full-length M(H)DM2 was due to binding and sequestration by an alternative-spliced M(H)DM2 product (HDM2ALT1). In another study, MDM2-D150-230 localized to the cytoplasm of U20S cells (Schuster et al., 2007, Mol. Cancer Res. 5:403-412 (“Schuster 2007”)). Both of these M(H)DM2 protein variants lacked part of the NH2-terminal region that contains a nuclear localization signal (NLS), suggesting that loss of this signal prevented the nuclear entry of these two proteins (Schuster 2007). Taken together, these data show that cellular localization of M(H)DM2 protein variants is highly complex.

Various M(H)DM4 protein variants have also been characterized, including splicing variant MDMX-S (Lenos and Jochemsen, 2011, J. Biomed Biotechnol., doi:10.1155/2011/876173).

HDM2 was found to be expressed in the plasma membrane of cancer cells (Sarafraz-Yazdi et al., 2010, PNAS 107:1918-1923 (“Sarafraz-Yazdi 2010”). Further, anti-cancer peptides, PNC-27 and PNC-28, which bind to HDM2 expressed in the cancer cell membranes and kill cancer cells by inducing necrosis, have been developed (Sarafraz-Yazdi 2010; Davitt et al., 2014, Annals Clin. Lab. Sci. 44:241248) (the amino acid sequences of PNC-27 and PNC-28 are provided in Table I of U.S. Patent Application Publication No. 2012/0177566). PNC-27 has been reported to bind within amino acids 25-109 of HDM2 (Do et al., 2003, Oncogene 22(10):1431-1444 (“Do 2003”); Chène, 2003, Nat. Rev. Cancer 3(2):102-109). Also, U.S. Patent Application Publication No. 2012/0177566 discloses methods of selectively necrosing cells by administering to the cells a compound (such as PNC-27 and PNC-28), including an HDM-2 targeting component and a cytotoxic component attached to the HDM-2 targeting component, wherein said compound comprises a membrane-active form. The membrane active function of PNC-27 and PNC-28 peptides (which comprise a membrane resident peptide (“MRP”) and a p53 sequence) is only achieved if the cargo (i.e., the p53 sequence component) is attached to the MRP component so as to form a cytotoxic structure (Kanovsky et al, 2001, PNAS 98:12438-12443 (“Kanovsky 2001”); Bowne et al., 2008, Ann Surg Oncol. 15:3588-3600 “Bowne 2008”)). When either the MRP component or the HDM-2 targeting component (i.e. p53 component) were used separately, they were found to be non-cytotoxic to cancer cells (Kanovsky 2001; Do 2003), demonstrating lack of activity of the MRP component alone and the HDM-2 targeting component alone. When recombinantly expressed inside the cell, the HDM-2 targeting component was observed to cause apoptosis (Bowne 2008). U.S. Pat. No. 9,765,117 discloses HDM2 targeting peptides and fusion peptides comprising an HDM2 targeting peptide and a transmembrane penetrating sequence, such as MRP; it is disclosed that MRP is required for induction of cell necrosis (see col. 4, lines 27-28). U.S. Pat. No. 9,765,117 indicates that expression of the p53 HDM2 targeting sequence in the absence of the MRP in cancer cells causes p53-dependent apoptosis and not tumor necrosis (see col. 4, lines 29-32).

The cell-penetrating peptides (“CPPs”) (such as MRPs, Membrane Transduction Domain of Antennapedia, trans-activating transcriptional activator (“TAT”) and Penetratin peptides) enable cellular membrane delivery of the peptides, and molecules attached to the peptides, to plasma membrane lipid bilayers, including those of normal healthy cells. These peptides were shown to efficiently transport various biologically active molecules inside living cells (Bechara et al., 2013, FEBS Lett. 587:1693-1702, Dupont et al., 2015, Methods Mol. Biol. 1324:29-37). The use of these CPPs (such as MRPs, Membrane Transduction Domain of Antennapedia, TAT and Penetratin peptides) linked to various cargos (such as other peptides, DNA, RNA, small molecules, antibodies or fragments thereof) have been demonstrated to improve pharmacokinetics, bio-distribution, retention, uptake and delivery of these cargos to various tumors in vitro and in vivo (Torchilin et al., 2003, PNAS 100:1972-1977; Shin et al., 2014, J. Biomed. Mater Res. A. 102:575-587; Kleemann et al., 2005, J. Control Release 109:299-316; Olson et al., 2009, Integr. Biol (Camb) 1:382-393; Jain et al., 2005, Cancer Res. 65:7840-7846; William et al., 2002, PNAS 99:10423-10428; Bolhassani, 2011, Biochim Biophys. Acta 1816:232-246). While most of these CPPs are used as cargo-delivery entities, Penetratin and MRP peptides have been reported to form a unique cytotoxic membrane-active structure upon linkage to their cargo (Rosal et al., 2005, Adv Drug Deliv Rev 57:653-60 (“Rosal 2005”); Bowne 2008; Kanovsky 2001). PNC-27 and PNC-28 peptides are examples of Penetratin-/MRP-cargo conjugates that exhibit a cytotoxic function that is dependent on the attachment and linkage of their cargo to the MRP, which is required for the formation of their membrane active structure, and hence, cytotoxic function (Kanovsky 2001; Rosal 2005; Bowne 2008).

There is long-standing unmet need in the art to effectively treat cancer, such as metastatic cancer, and to prevent metastasis in patients.

5. SUMMARY OF THE INVENTION

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4. In particular, described herein are antibodies or fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibodies or fragments inhibit tumor growth in vivo (or inhibit tumor cell proliferation in vivo). In certain embodiments, the antibodies or fragments described herein are not bound to a cell-penetrating peptide (e.g., a membrane resident peptide). In certain embodiments, the antibodies or fragments described herein are not bound to a cytotoxic component (i.e., not bound to a cytotoxic agent).

In one aspect, described herein are antibodies or fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In particular, described herein are antibodies or fragments that specifically bind to M(H)DM2/4 (e.g., HDM2), wherein the antibody or fragment specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, the antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1). In one embodiment, the antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is TTSQIPASEQE (SEQ ID NO:2). In one embodiment, the antibody or fragment binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In certain embodiments, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1. In one embodiment, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:2. In one embodiment, described herein is an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:3.

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising: (i) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR3; said VH CDR 1, VH CDR 2 and VH CDR 3 being the CDRs of a VH that has an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40, or (ii) a light chain variable region (VL) comprising VL CDR 1, VL CDR 2 and VL CDR 3 being the CDRs of a VL that has an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:41. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:36. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:38. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:40. In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:37 (and, optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:36). In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:39 (and, optionally, a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:38). In one embodiment, the humanized antibody or a fragment that specifically binds to HDM2 comprises a VL wherein VL CDR 1, VL CDR 2 and VL CDR 3 are of a VL having the amino acid sequence of SEQ ID NO:41 (and, optionally, the a VH wherein VH CDR 1, VH CDR 2 and VH CDR 3 are of a VH having the amino acid sequence of SEQ ID NO:40).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to M(H)DM2/4, said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to M(H)DM2/4 (e.g., HDM2), said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO:52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58); or (v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).

In one aspect, provided herein is an antibody or a fragment thereof that specifically binds to M(H)DM2/4 (e.g., HDM2), said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:

(i) the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (iii) the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein:

(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO:22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO:50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51); or

(iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO:147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23). In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein:

(i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); or (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51).

In one aspect, described herein is an antibody or a fragment comprising:

(a) a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); or (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51); and (b) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146); and (b) a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO:22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO:50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51); or (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO:147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO:52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58); or (v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:

(i) the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); or (ii) the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a light chain variable region (VL) comprising a VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein:

(i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO:27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO:28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO:59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO:60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO:61); or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO:141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO:52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58); or (v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58); and (b) a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO:27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO:28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO:59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO:60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO:61); or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO:141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein:

(i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising a VH, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR 3, wherein:

(i) the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (ii) the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).

In one aspect, described herein is an antibody or fragment further comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein:

(i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO:33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO:34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO:35); or (ii) the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO:69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO:70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO:71).

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68); and (b) a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO:33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO:34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO:35); or (ii) the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO:69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO:70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO:71).

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:36, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:36.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:37, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:37 (and, optionally, comprising a VH having the amino acid sequence of SEQ ID NO:36, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:36).

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:38, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:38.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:39, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:39 (and, optionally, comprising a VH having the amino acid sequence of SEQ ID NO:38, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:38).

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH having the amino acid sequence of SEQ ID NO:40, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:40.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VL having the amino acid sequence of SEQ ID NO:41, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:41 (and, optionally, comprising a VH having the amino acid sequence of SEQ ID NO:40, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:40).

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, or a VH having at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and/or (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311, or a VL having at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311.

In one aspect, described herein is a humanized antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (ii) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:287, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:287.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:291, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:291.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:295, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:295.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:299, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:299.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:305, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:305.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:309, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:309.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:289, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:289.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:293, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:293.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:297, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:297.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:301, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:301.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:303, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:303.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:307, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:307.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:311, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:311.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:299, and (ii) a VL having the amino acid sequence of SEQ ID NO:297.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, and SEQ ID NO:299, and (ii) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, and SEQ ID NO:303.

In one aspect, described herein is a humanized antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (ii) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:297, SEQ ID NO:307, and SEQ ID NO:311.

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO:283, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:283, and (b) a light chain variable region (VL) having an amino acid sequence of SEQ ID NO:285, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:285.

In one aspect, described herein is an antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO:283, and (b) a light chain variable region (VL) having an amino acid sequence of SEQ ID NO:285.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VH having the amino acid sequence of SEQ ID NO:283, or a VH having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:283.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a VL having the amino acid sequence of SEQ ID NO:285, or a VL having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:285.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2, said antibody comprising:

(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, or a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:312, and/or (b) a light chain having an amino acid sequence of SEQ ID NO:313, or a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:313.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2, said antibody comprising:

(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, and (b) a light chain having an amino acid sequence of SEQ ID NO:313, or a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:313.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a heavy chain having the amino acid sequence of SEQ ID NO:312, or a heavy chain having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:312.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2 comprising a light chain having the amino acid sequence of SEQ ID NO:313, or a light chain having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 313.

In one aspect, described herein is an antibody or fragment thereof that specifically binds to HDM2 or MDM2, which inhibits tumor cell proliferation in vivo.

In preferred embodiments, the anti-M(H)DM2/4 antibody described herein is a monoclonal antibody. In certain embodiments, the anti-M(H)DM2/4 antibody described herein is a human, humanized, or a chimeric antibody (e.g., a human, humanized or chimeric monoclonal antibody). In one embodiment, the anti-M(H)DM2/4 antibody described herein is a human antibody. In one embodiment, the anti-M(H)DM2/4 antibody described herein is a humanized antibody. In one embodiment, the anti-M(H)DM2/4 antibody described herein is a chimeric antibody.

In certain embodiments, the anti-M(H)DM2/4 antibody described herein is a purified antibody.

In certain embodiments, the anti-M(H)DM2/4 antibody described herein is an immunoglobulin (e.g., IgG or IgM). In one embodiment, the immunoglobulin is an IgG. In one embodiment, the immunoglobulin is an IgM. In certain embodiments, the immunoglobulin is of IgG1 isotype. In other embodiments, the immunoglobulin is of IgG3 isotype. In other embodiments, the immunoglobulin is of IgG2 isotype. In other embodiments, the immunoglobulin is of IgG4 isotype. In certain embodiments, the anti-M(H)DM2/4 antibody described herein comprises an Fc region, wherein the Fc region is a human IgG Fc region or a human IgM Fc region. In specific embodiments, the anti-M(H)DM2/4 antibody described herein comprises an Fc region, which is a human IgG1 Fc region, a human IgG2 Fc region, or a human IgG3 Fc region, or a human IgG4 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG1 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG3 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG4 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgG2 Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgM Fc region. In one embodiment, the anti-M(H)DM2/4 antibody described herein comprises a human IgE Fc region.

In certain embodiments, the anti-M(H)DM2/4 antibody or fragment described herein is an antigen-binding fragment of an anti-M(H)DM2/4 antibody. In certain embodiments, the antibody or fragment described herein is an Fv fragment, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a single chain antibody molecule, or a single chain Fv (scFv). In one embodiment, the antibody or fragment described herein is an Fv fragment. In one embodiment, the antibody or fragment described herein is a Fab fragment. In one embodiment, the antibody or fragment described herein is a Fab′ fragment. In one embodiment, the antibody or fragment described herein is a F(ab′)2 fragment. In one embodiment, the antibody or fragment described herein is a single chain antibody molecule. In one embodiment, the antibody or fragment described herein is a single chain Fv (scFv).

In certain embodiments, the anti-M(H)DM2/4 antibody or antigen-binding fragment described herein mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytoxicity (ADCC). In one embodiment, the anti-M(H)DM2/4 antibody or antigen-binding fragment mediates complement-dependent cytotoxicity (CDC).

In certain embodiments, the anti-M(H)DM2/4 antibody described herein is a bispecific antibody that also specifically binds to a cell surface antigen of an effector cell (e.g., a T cell, a B lymphocyte, a neutrophil, a macrophage, a natural killer cell, or a dendritic cell).

In one embodiment, the antibody or fragment described herein specifically binds to an extracellularly accessible epitope of M(H)DM2 (e.g., HDM2) and does not bind to M(H)DM4 (e.g., HDM4). In another embodiment, the antibody or fragment described herein specifically binds to extracellularly accessible epitopes of both M(H)DM2 (e.g., HDM2) and M(H)DM4 (e.g., HDM4). In one embodiment, the antibody or fragment described herein specifically binds to an extracellularly accessible epitope of HDM2, and optionally, may also bind to an extracellularly accessible epitope of MDM2.

In a specific embodiment, the anti-HDM2 antibody or fragment described herein specifically binds HDM2 within amino acids of SEQ ID NO: 1 (which are amino acids 1 to 15 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody described herein specifically binds HDM2 within amino acids of SEQ ID NO: 2 (which are amino acids 15 to 25 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody described herein specifically binds HDM2 within amino acids of SEQ ID NO: 3 (which are amino acids 475-491 of HDM2 (SEQ ID NO:4)). In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 19 to 50 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 154 to 167 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 60 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 100 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 100 to 110 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 50 to 60 of SEQ ID NO: 4. In yet another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 1 to 109 of SEQ ID NO: 4. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within amino acids 26 to 60 of SEQ ID NO: 4. In one specific embodiment, the anti-HDM2 antibody described herein specifically binds within the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells. In another specific embodiment, the anti-HDM2 antibody described herein specifically binds within the terminal 100 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells.

In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind to the epitope of HDM2 or MDM2 to which “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) binds. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not compete with “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) for binding to HDM2.

In another embodiment, the anti-M(H)DM2/4 antibody described herein binds within amino acids 101 to 200 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein binds to the epitope of HDM2 or MDM2 to which “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) binds. In one embodiment, the anti-M(H)DM2/4 antibody described herein competes with “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) for binding to HDM2.

In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 153 to 222 of SEQ ID NO:4. In one embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 26 to 169 of SEQ ID NO:4. In a specific embodiment, the anti-M(H)DM2/4 antibody described herein does not bind within amino acids 26 to 222 of SEQ ID NO:4.

In certain embodiments, the M(H)DM2/4 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an M(H)DM2/4 variant that lacks one or more nuclear localization signal domains. In specific embodiments, the HDM2 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an HDM2 variant that lacks the sequence of amino acids 179 to 185 of SEQ ID NO: 4 and/or the sequence of amino acids 464 to 471 of SEQ ID NO: 4. In one embodiment, the HDM2 exposed on the surface of cancer cells being targeted by the antibodies or fragments described herein is an HDM2 variant that lacks the sequence of amino acids 181 to 185 of SEQ ID NO: 4.

In one aspect, provided herein are antibodies or fragments thereof that compete for binding to M(H)DM2/4 with an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment thereof described herein. Preferably, such antibodies or fragments that compete for binding are monoclonal antibodies or fragments thereof.

In one aspect, provided herein are antibodies or fragments thereof that compete for binding to M(H)DM2/4 with a mouse anti-HDM2 immunoglobulin (preferably IgG) antibody selected from the group consisting of: (i) an antibody comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:36, and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:37; (ii) an antibody comprising a VH having the amino acid sequence of SEQ ID NO:38, and a VL having the amino acid sequence of SEQ ID NO:39; and (iii) an antibody comprising a VH having the amino acid sequence of SEQ ID NO:40, and a VL having the amino acid sequence of SEQ ID NO:41.

In one aspect, provided herein are antibodies or fragments thereof that: (i) compete for binding to a peptide of sequence SEQ ID NO:1 with a mouse anti-HDM2 IgG1 antibody comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:36, and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:37; or (ii) compete for binding to a peptide of SEQ ID NO:2 with a mouse anti-HDM2 IgG3 antibody comprising a VH having the amino acid sequence of SEQ ID NO:38, and a VL having the amino acid sequence of SEQ ID NO:39; or (iii) compete for binding to a peptide of SEQ ID NO:3 with a mouse IgM antibody comprising a VH having the amino acid sequence of SEQ ID NO:40, and a VL having the amino acid sequence of SEQ ID NO:41.

In one aspect, provided herein are antibody-drug conjugates comprising any antibody or fragment described herein (e.g., an antibody-drug conjugate in which an anti-M(H)DM2/4 antibody or fragment thereof described herein is covalently bound to a cytotoxic drug).

In one aspect, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of any antibody or fragment described herein.

In one aspect, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.

In one aspect, provided herein are methods for treating cancer in a subject in need thereof, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide); or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein are methods for treating cancer in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein are methods for treating cancer in a subject in need thereof, said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for treating cancer in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for treating cancer in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In one embodiment, provided herein is a method for treating cancer in a subject in need thereof, said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein. In one aspect, provided herein are methods for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein are methods for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein are methods for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetraitng peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, provided herein is a method for inhibiting tumor growth in a subject in need thereof, said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein. In one aspect, provided herein are methods for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein are methods for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein are methods for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for inhibiting tumor progression in a subject in need thereof, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In one aspect, provided herein is a method for preventing cancer recurrence or relapse in a subject in need thereof (e.g., a subject who is in remission from cancer), said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for preventing cancer recurrence or relapse in a subject in need thereof (e.g., a subject who is in remission from cancer), said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein is a method for preventing cancer recurrence or relapse in a subject in need thereof (e.g., a subject who is in remission from cancer), said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for preventing cancer recurrence or relapse in a subject in need thereof (e.g., a subject who is in remission from cancer), said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for preventing cancer recurrence or relapse in a subject in need thereof (e.g., a subject who is in remission from cancer), said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, provided herein is a method for preventing cancer recurrence or relapses in a subject in need thereof, said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.

In one aspect, provided herein is a method for increasing survival in a subject having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein is a method for increasing survival in a subject having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein is a method for increasing survival in a subject a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for increasing survival in a subject having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for increasing survival in a subject having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment), said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, provided herein is a method for increasing survival in a subject having a cancer (e.g., relative to a subject not treated with anti-M(H)DM2/4 antibody or fragment thereof described herein), said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.

In one aspect, provided herein are methods for preventing metastasis in a subject

having a cancer, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein are methods for preventing metastasis in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein are methods for preventing metastasis in a subject having a cancer, said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for preventing metastasis in a subject having a cancer, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for preventing metastasis in a subject having a cancer, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, provided herein is a method for preventing metastasis in a subject having a cancer, said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein. In one aspect, provided herein are methods for inhibiting metastasis (e.g., reducing the number, size or invasiveness of metastases) in a subject having a metastatic cancer, said method comprising administering to the subject: (i) any anti-M(H)DM2/4 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one aspect, provided herein are methods for inhibiting metastasis in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component. In one aspect, provided herein are methods for inhibiting metastasis (e.g., reducing the number, size or invasiveness of metastases) in a subject having a metastatic cancer, said method comprising administering to the subject: (i) any anti-HDM2 antibody or fragment described herein; (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2, wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) any pharmaceutical composition described herein, or (iv) any antibody-drug conjugate described herein. In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size or invasiveness of metastases) in a subject having a metastatic cancer, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In one embodiment, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size or invasiveness of metastases) in a subject having a metastatic cancer, said method comprising administering to the subject an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 contained within a peptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, provided herein is a method for inhibiting metastasis (e.g., reducing the number, size or invasiveness of metastases) in a subject having a metastatic cancer, said method comprising administering to the subject any chimeric or humanized anti-M(H)DM2/4 antibody or fragment described herein.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, said method comprising administering to the subject:

(i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) any anti-M(H)DM2/4 antibody or fragment described herein, (iv) an antibody-drug conjugate comprising any anti-M(H)DM2/4 antibody or fragment described herein, bound to a cytotoxic drug, or (v) a pharmaceutical composition comprising a therapeutically effective amount of any anti-M(H)DM2/4 antibody or fragment described herein or an antibody-drug conjugate comprising any anti-M(H)DM2/4 antibody or fragment described herein, bound to a cytotoxic drug.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, wherein the one or more inhibitory checkpoint molecules are selected from the group consisting of: CTLA-4, PD-1, PD-L1, and PD-L2.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, wherein the one or more inhibitory checkpoint molecules is PD-1.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, wherein the inhibitor of one or more inhibitory checkpoint molecules is an inhibitory antibody to PD-1.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, wherein said method comprises administering to the subject any anti-M(H)DM2/4 antibody or fragment described herein.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, wherein said method comprises administering to the subject any anti-M(H)DM2/4 antibody or fragment described herein, wherein the anti-M(H)DM2/4 antibody or fragment is not bound to a cell-penetrating peptide.

In one aspect, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, said methods comprising administering to the subject any pharmaceutical composition comprising a therapeutically effective amount of any anti-M(H)DM2/4 antibody or fragment described herein or an antibody-drug conjugate comprising any anti-M(H)DM2/4 antibody or fragment described herein, bound to a cytotoxic drug, wherein the antibody or fragment is not bound to a cell-penetrating peptide.

In one aspect, provided herein is any method described herein, wherein the method comprises administering an anti-M(H)DM2/4 antibody or fragment which competes for binding to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds to HDM2 or MDM2, said humanized antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, and SEQ ID NO:299, and (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, and SEQ ID NO:303.

In one aspect, provided herein is any method described herein, wherein the method comprises administering an anti-M(H)DM2/4 antibody or fragment which competes for binding to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds to HDM2 or MDM2, said humanized antibody or fragment comprising:

(a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID SEQ ID NO:297, SEQ ID NO:307, and SEQ ID NO:311.

In one aspect, provided herein is any method described herein, wherein the method comprises administering an anti-M(H)DM2/4 antibody or fragment which competes for binding to M(H)DM2/4 with an antibody or a fragment that specifically binds to HDM2 or MDM2, said antibody or fragment comprising:

(a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO:283, and (b) a light chain variable region (VL) having an amino acid sequence of SEQ ID NO:285.

In one aspect, provided herein are any method described herein, wherein the method comprises administering an anti-M(H)DM2/4 antibody or fragment which competes for binding to M(H)DM2/4 with a chimeric antibody that specifically binds to HDM2 or MDM2, said chimeric antibody comprising:

(a) a heavy chain having an amino acid sequence of SEQ ID NO:312, and (b) a light chain having an amino acid sequence of SEQ ID NO:313.

In one aspect, provided herein is any method described herein, wherein the anti-M(H)DM2/4 antibody or fragment is purified.

In one aspect, provided herein are any method described herein, wherein the anti-M(H)DM2/4 antibody or fragment is not bound to a cytotoxic drug.

In one aspect, provided herein are methods of selecting and treating a subject (e.g., a human) having a cancer, said method comprising: (a) identifying a subject having a cancer wherein an antibody or a fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) binds to the surface of an intact cell of the cancer; and (b) administering to the subject (i) any M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein or an antibody-drug conjugate comprising said antibody or fragment (e.g., an anti-M(H)DM2/4 antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide consists of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3); (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cell-penetrating peptide (e.g., a membrane resident peptide), or an antibody-drug conjugate comprising the antibody or fragment (i.e., said antibody or fragment that is not bound to a cell-penetrating peptide) bound to a cytotoxic drug, (iii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibody or fragment is not bound to a cytotoxic component, (iv) any pharmaceutical composition described herein, or (v) any antibody-drug conjugate described herein. The antibody or fragment thereof in step (b) can be the same or different from the antibody or fragment thereof in step (a). In certain embodiments, provided herein are methods that further comprise, before step (b), a step of determining whether the antibody or fragment binds to the surface of the intact cell of the cancer (e.g., using FACS or cell-based ELISA analysis) (using any anti-M(H)DM2/4 antibody described herein). In one embodiment, provided herein are methods that further comprise, before the determining step, the step of obtaining intact cells of the cancer (e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject). In one embodiment, the method comprises administering to the subject an antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.

In one aspect, provided herein are methods for selecting a subject (e.g., a human) for treatment and treating cancer in the subject, said method comprising: (a) selecting a subject having a cancer for treatment by: (i) obtaining an intact cancer cell from the subject (e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject), and (ii) determining whether an antibody or a fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2/4 (e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, such as any anti-M(H)DM2/4 antibody or fragment described herein) binds to the surface of the intact cancer cell obtained from the subject (e.g., using FACS or cell-based ELISA analysis), and (b) if the binding is detected in step (a), administering to the subject said antibody or fragment, wherein said antibody or fragment is not bound to a cell-penetrating peptide. In one aspect, provided herein are methods for selecting a subject for treatment and treating cancer in the subject, said method comprising: (a) selecting a subject having a cancer for treatment by: (i) obtaining an intact cancer cell from the subject (e.g., by biopsy of the cancerous tumor in the subject, or by obtaining a blood sample with circulating cancer cells from the subject), and (ii) determining whether an antibody or a fragment thereof that specifically binds to HDM2 (e.g., an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2, such as any anti-M(H)DM2/4 antibody or fragment described herein) binds to the surface of the intact cancer cell obtained from the subject (e.g., using FACS or cell-based ELISA analysis), and (b) if the binding is detected in step (a), administering to the subject said antibody or fragment, wherein said antibody or fragment is not bound to a cell-penetrating peptide.

In certain aspects, provided herein are methods of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, said method comprising:

(a) identifying a subject who (i) has experienced accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, and (ii) has a cancer wherein an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer; and (b) administering to the subject: (i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) any anti-M(H)DM2/4 antibody or fragment described herein, (iv) any antibody-drug conjugate described herein, or (v) any pharmaceutical composition described herein. In certain embodiments of the methods, the method further comprises before step (b), a step of determining whether any antibody or fragment thereof described herein that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer. In certain embodiments of the method, step (a) further comprises selecting a subject who has a gene amplification of M(H)DM2/4. In certain embodiments of the methods, step (a) further comprises selecting a subject who has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells. In certain embodiments of the methods, step (a) further comprises selecting a subject who has a cancer wherein there is an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to the binding of the antibody or fragment thereof to the surface of intact normal cells.

In certain aspects, provided herein are methods of treating cancer in a subject who is a hyper-progressor in response to administration of an inhibitor of one or more inhibitory checkpoint molecules, said methods comprising: (a) identifying a subject, wherein the subject has (i) a cancer wherein an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer, and (ii) a gene amplification of M(H)DM2/4, or an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells; and (b) administering to the subject: (i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) any anti-M(H)DM2/4 antibody or fragment thereof described herein, (iv) any antibody-drug conjugate described herein, or (v) any pharmaceutical composition described herein. In certain embodiments of the methods, the method further comprises before step (b) a step of determining whether the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer. In certain embodiments of the methods, step (a) further comprises selecting a subject who has a gene amplification of M(H)DM2/4. In certain embodiments of the methods, step (a) further comprises selecting a subject who has an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to its binding to the surface of intact normal cells.

In certain embodiments, the cancer treated in accordance with the methods described herein is a type of cancer that is known to metastasize. In some embodiments, the cancer treated in accordance with the methods described herein is an advanced stage cancer. In other embodiments, the cancer treated in accordance with the methods described herein is an early stage cancer. In specific embodiments, the cancer treated in accordance with the methods described herein is a metastatic cancer. The cancer being treated can be a solid cancer or a non-solid cancer (e.g., leukemia or lymphoma).

In certain embodiments, the cancer treated in accordance with the methods described herein is a cervical cancer, an endometrial cancer, an ovarian cancer, a pancreatic cancer, a melanoma (e.g., a uveal melanoma), a breast cancer, a triple negative breast cancer, a colorectal cancer (e.g. a colon cancer), a bladder cancer, an astrocytic neoplasm, a glioblastoma, a pediatric Rhabdomyosarcoma, or a lung cancer (e.g., a non-small cell lung carcinoma). In specific embodiments, the cancer treated in accordance with the methods described herein is a melanoma, a pancreatic cancer, a breast cancer, or an ovarian cancer. In one embodiment, the cancer treated in accordance with the methods described herein is a lung cancer. In one embodiment, the cancer treated in accordance with the methods described herein is a colorectal cancer. In one embodiment, the cancer treated in accordance with the methods described herein is a colon cancer. In one embodiment, the cancer treated in accordance with the methods described herein is a melanoma. In one embodiment, the cancer treated in accordance with the methods described herein is a pancreatic cancer. In one embodiment, the cancer treated in accordance with the methods described herein is a breast cancer. In one embodiment, the cancer treated in accordance with the methods described herein is an ovarian cancer.

The subject treated in accordance with the methods described herein can be a human or a non-human animal (such as a mammal). In a preferred embodiment, the subject is a human.

In certain embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or intratumorally. In other embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are administered orally.

In certain embodiments, the subject being treated in accordance with the methods described herein is further administered an additional anti-cancer therapy that is different from said antibody or fragment or antibody-drug conjugate (e.g., vaccine, targeted therapy, chemotherapy, radiotherapy, surgery, or immunotherapy). In one embodiment, the additional therapy is a vaccine. In one embodiment, the additional therapy is a targeted therapy. In one embodiment, the additional therapy is a chemotherapy (e.g., gemcitabine, paclitaxel, nab-paclitaxel, or a combination of gemcitabine and nab-paclitaxel). In one embodiment, the additional therapy is an immunotherapy. In one embodiment, the additional therapy is a radiotherapy. In one embodiment, the additional therapy is a surgery (e.g., to remove part or all of the cancerous tumor being treated). In specific embodiments, the additional therapy is an inhibitor of the function of one or more checkpoint inhibitory molecules (e.g., an inhibitor, such as an inhibitory antibody to, one or more of: CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, OX40, and LAG-3). In certain embodiments, the additional therapy is not a cell cycle inhibitor. In a specific embodiment, the subject treated using the methods described herein is not administered a cell cycle inhibitor during the course of treatment with said antibody or fragment. In certain embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are administered alone, without any additional anti-cancer therapy (e.g., the subject treated using the methods described herein is not administered an additional anti-cancer therapy during the course of treatment with said antibody or fragment).

In certain embodiments, the subject being treated in accordance with the methods described herein is further administered an additional cancer therapy that is different from an anti-M(H)DM2/4 antibody or fragment or antibody-drug conjugate, wherein the additional cancer therapy is an inhibitor of one or more of: EGFR, KRAS, STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK, PI3K/AKT, TGF-B, DNMT3A, IFN□, JAK1/JAK2/JAK3, CD274, PTEN, ART, AND CDK.

In certain embodiments, the subject being treated in accordance with the methods described herein is further administered an additional cancer therapy that is different from an anti-M(H)DM2/4 antibody or fragment or antibody-drug conjugate, wherein the additional cancer therapy is a peptide inhibitor of p53-M(H)DM2/4 interaction.or a small molecule inhibitor of p53-M(H)DM2/4 interaction.

In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is cisplatin. In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is 5-FU. In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is paclitaxel. In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is paclitaxel formulated as albumin-bound particles (e.g., ABRAXANE®). In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is gemcitabine (e.g., where the cancer being treated is a pancreatic cancer). In certain embodiments, the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is nab-paclitaxel (e.g., where the cancer being treated is a pancreatic cancer). In certain embodiments, the cancer is a pancreatic cancer, and the subject being treated in accordance with the methods described herein is further administered chemotherapy, wherein the chemotherapy is a combination of gemcitabine and nab-paclitaxel. In certain embodiments, the gemcitabine and/or nab-paclitaxel are administered in doses that are lower than doses used when gemcitabine and/or nab-paclitaxel are administered not in combination with an anti-cancer antibody (such as an anti-M(H)DM2/4 antibody or fragment described herein). In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose that is less than 1,500 mg/m², and/or nab-paclitaxel is administered in a dose that is less than 300 mg/m². In one embodiment, wherein the subject is human, gemcitabine is administered in a dose that is equal to or less than 1,000 mg/m² and/or the nab-paclitaxel is administered in a dose that is equal to or less than 125 mg/m². In one embodiment, wherein the subject is human, gemcitabine is administered in a dose that is equal to or less than 500 mg/m² and/or the nab-paclitaxel is administered in a dose that is equal to or less than 62.5 mg/m². In certain embodiments, the combination of gemcitabine and nab-paclitaxel is administered with a frequency of every 2 weeks or less.

In certain embodiments, the subject being treated in accordance with the methods described herein is resistant to other cancer therapies (e.g., vaccine, targeted therapy, chemotherapy, radiotherapy, surgery, or immunotherapy). In specific embodiments, the subject being treated in accordance with the methods described herein is resistant to chemotherapy. In one embodiment, the subject being treated in accordance with the methods described herein has a chemotherapy-resistant ovarian cancer. In other embodiments, the subject being treated in accordance with the methods described herein is resistant to one or more inhibitor of an inhibitory immune checkpoint molecule. In other embodiments, the subject being treated in accordance with the methods described herein is resistant to radiotherapy.

In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein specifically binds within amino acids 19 to 50 of SEQ ID NO:4. In another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 154 to 167 of SEQ ID NO:4. In yet another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 1 to 60 of SEQ ID NO:4. In yet another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 1 to 100 of SEQ ID NO:4. In yet another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 1 to 109 of SEQ ID NO:4. In another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 26 to 60 of SEQ ID NO: 4. In one specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of cancer cells. In another specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within the terminal 100 amino acids at the C-terminus of the HDM2 on the plasma membrane of cancer cells. In one specific embodiment, the anti-M(H)DM2/4 antibody used in the methods described herein specifically binds within amino acids 101 to 200 of SEQ ID NO:4.

In particular embodiments, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody OP145 (which is described herein, see, e.g., Table 10). In other particular embodiments, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody 965 (SMP14) (which is described herein, see, e.g., Tables 3 and 10). In yet other particular embodiments, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-813 (N-20) (which is described herein, see, e.g., Table 10). In another embodiment, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-812 (C-18) (which is described herein, see, e.g., Table 10). In another embodiment, the anti-M(H)DM2/4 antibody or fragment used in the methods described herein competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody M01, clone 1A7 (which is described herein, see, e.g., Table 3).

In certain aspects, provided herein are methods for treating cancer or preventing metastases in a subject in need thereof, said method comprising administering to the subject any anti-M(H)DM2/4 antibody described herein (such as an antibody that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 on the surface of cells of said cancer), wherein the antibody comprises a human IgG Fc region that mediates complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC). In some of these embodiments, the extracellular region of HDM2 targeted by the anti-HDM2 antibodies or fragments used herein is within one of the following amino acid regions of HDM2: amino acids of SEQ ID NO: 1 (which are amino acids 1 to 15 of SEQ ID NO:4), amino acids of SEQ ID NO: 2 (which are amino acids 15 to 25 of SEQ ID NO:4), amino acids of SEQ ID NO: 3 (which are amino acids 475 to 491 of SEQ ID NO:4), amino acids 19 to 50 of SEQ ID NO: 4, amino acids 50 to 60 of SEQ ID NO: 4, amino acids 100 to 110 of SEQ ID NO: 4, amino acids 154 to 167 of SEQ ID NO: 4, amino acids 1 to 60 of SEQ ID NO: 4, or the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells. In specific embodiments of the methods described in this paragraph, the cancer is a leukemia, a lung cancer, a colon cancer, a melanoma, a pancreatic cancer, a breast cancer, or an ovarian cancer.

In one aspect, provided herein are methods of diagnosing cancer in a subject (e.g., a human), said method comprising: (a) detecting whether an antibody or a fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2/4 (e.g., HDM2) binds to the surface of intact cells of the subject, wherein the antibody or fragment is any anti-M(H)DM2/4 antibody or fragment described herein (in a preferred example, wherein the antibody or fragment is any anti-M(H)DM2/4 antibody or fragment that specifically binds to a peptide of SEQ ID NO″1, SEQ ID NO:2, or SEQ ID NO:3); and (b) diagnosing the subject with cancer if binding is detected in step (a). In one embodiment, the method of diagnosing is an ex vivo method. In one embodiment, the method of diagnosing further comprises, before step (a), obtaining intact cells from the subject. In one embodiment, the method of diagnosing comprises administering the antibody or fragment to the subject before the detecting in step (a), and wherein the detecting is performed by in vivo imaging of the subject.

In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, said method comprising: (a) determining whether a gene amplification of M(H)DM2/4 is present in the cells of the cancer of the subject; (b) determining whether an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the subject; and (c) diagnosing the subject with the hyper-progressive disease if the gene amplification is determined to be present in step (a) and binding is detected in step (b). In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, said methods are ex vivo methods. In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, said methods further comprising obtaining intact cells from the subject before step (b). In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, said methods comprising administering the antibody or fragment to the subject before the detecting in step (b), and wherein the detecting is performed by in vivo imaging of the subject. In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, wherein, in step (b), the antibody or fragment is labeled. In certain aspects, provided herein are methods of diagnosing a hyper-progressive disease in a subject who has cancer, wherein any anti-M(H)DM2/4 antibody or fragment thereof described herein is used. In certain aspects of the methods, the subject is a human.

In one aspect, provided herein are vaccine compositions comprising: (i) an immunogenic amount of a peptide, wherein the amino acid sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3), or a polynucleotide encoding the peptide; and (ii) a pharmaceutically acceptable carrier. In certain aspects, the peptide in the vaccine compositions is purified. In certain aspects, vaccine compositions further comprise an adjuvant. In one aspect, provided herein are methods of vaccinating a subject at risk for developing cancer or a subject who has been diagnosed with cancer by administering to the subject the vaccine composition described herein.

5.1 Terminology

As used herein, the term “HDM2” refers to the human E3 ubiquitin-protein ligase of UniProt Accession Number Q00987 (SEQ ID NO:4) (i.e., full-length HDM2 protein) or a protein product of any splice variant of the full-length HDM2 protein known in the art or described herein. The amino acid sequences of exemplary splice variants of the full-length HDM2 protein are shown as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID N011, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

As used herein, the term “MDM2” refers to the mouse E3 ubiquitin-protein ligase of UniProt Accession Number P23804 (SEQ ID NO:5) (i.e., full-length MDM2 protein) or a protein product of any splice variant of the full-length MDM2 protein known in the art or described herein.

As used herein, the term “M(H)DM2” refers to HDM2, MDM2, or an E3 ubiquitin-protein ligase from species other than human and mouse that is a homolog of HDM2 or MDM2.

As used herein, the term “HDM4” refers to the human protein of UniProt Accession Number O15151 (SEQ ID NO:6) (i.e., full-length HDM4 protein) or a protein product of any splice variant of the full-length HDM4 protein known in the art or described herein.

As used herein, the term “MDM4” refers to the mouse protein of UniProt Accession Number O35618 (i.e., full-length MDM4 protein) or a protein product of any splice variant of the full-length MDM4 protein known in the art or described herein. The amino acid sequence of an exemplary splice variant of the full-length MDM4 protein is shown as SEQ ID NO:6. Other splice variants of the full length MDM4 protein known in the art include, without limitation MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211.

As used herein, the term “M(H)DM4” refers to HDM4 (also called HDMX), MDM4 (also called MDMX), or a protein from a species other than human and mouse that is a homolog of HDM4 or MDM4.

As used herein, the term “M(H)DM2/4” refers to HDM2, MDM2, HDM4, MDM4, or a protein from a species other than human and mouse that is a homolog of HDM2, MDM2, HDM4 or MDM4.

As used herein, the term “about,” when used to modify a numeric value, indicate that deviations of up to 10% above and below the numeric value remain within the intended meaning of the recited value.

As used herein, the term “intact” with reference to a cell refers to a cell that is viable or fixed but not permeabilized.

As used herein, the term “extracellularly accessible” with reference to an epitope of M(H)DM2/4, refers to an epitope of M(H)DM2/4 that, when the M(H)DM2/4 is expressed by an intact cell, the epitope is available for binding with an extracellular antibody (without a need for intracellular transport of the antibody). An antibody or a fragment thereof can be determined to bind to an extracellularly accessible epitope of M(H)DM2/4, when the antibody, when extracellular, binds to M(H)DM2/4 expressed by an intact cell. As will be clear to one skilled in the art, the M(H)DM2/4 expressed by an intact cell in the foregoing definition is the form of the M(H)DM2/4 retained on the plasma membrane (i.e., as a transmembrane protein).

“Membrane-bound M(H)DM2/4” or “plasma membrane-bound M(H)DM2/4” refers to a transmembrane M(H)DM2/4 protein variant.

As used herein, the term “VL” refers to the light chain variable region of an antibody.

As used herein, the term “VK” refers to the kappa isotype of the light chain variable region of an antibody.

As used herein, the term“VH” refers to the heavy chain variable region of an antibody.

As used herein, the term “hyper-progression” refers to an accelerated rate of tumor or cancer growth therapy after administration of a therapy compared to prior to the administration of the therapy.

As used herein, the term “hyper-progressor” refers to a subject who exhibits an accelerated rate of tumor or cancer growth after administration of a therapy compared to prior to the administration of the therapy.

As used herein, the term “percent (%) amino acid sequence identity” or “percent sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known in the art, for instance, using publicly available computer software such as BLASTp, BLAST-2, ALIGN (e.g., ALIGN-2) or Megalign (DNASTAR) software.

6. BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-D show that monoclonal antibodies NMC-103, NMC-204 and NMC-303 specifically bound to NMC-P1 (SEQ ID NO:1), NMC-P2 (SEQ ID NO:2) and NMC-P3 (SEQ ID NO:3) peptide antigens, respectively, in peptide-ELISA experiments. (A) NMC-103 bound to NMC-P1 peptide while NMC-204 did not show binding to NMC-P1. (B) NMC-204 bound to NMC-P2 peptide while NMC-103 did not bind to NMC-P2. (C) NMC-303 bound to NMC-P3 peptide while NMC-204 did not bind to NMC-P3. (D) While NMC-103 bound to NMC-P1, pre-incubation of NMC-P1 peptide with NMC-103 abolished the binding of NMC-103 to NMC-P1 peptide on the plate. In contrast, pre-incubation of NMC-103 with either NMC-P2 or NMC-P3 did not affect the binding of NMC-103 to NMC-P1.

FIG. 2 shows that monoclonal antibodies NMC-103, NMC-204, NMC-303 bound to HDM2 recombinant protein.

FIGS. 3A-B show that monoclonal antibody NMC-103 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.

FIGS. 4A-B show that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.

FIGS. 5A-B show that monoclonal antibody NMC-303 bound to an extracellularly accessible epitope of HDM2 on intact human (A) and murine (B) cancer cells.

FIG. 6 shows that monoclonal antibody NMC-204 bound to an extracellularly accessible epitope of HDM2 on intact human cancer cells but did not bind to intact normal human peripheral blood mononuclear cells.

FIGS. 7A-B depict the binding curves of the binding of monoclonal antibodies NMC-103 (A) and NMC-204 (B) to intact MIA PaCa-2 cells.

FIGS. 8A-C show that the binding of monoclonal antibody NMC-103 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIA PaCa-2 cells was competed by the NMC-P1 peptide (A), the binding of monoclonal antibody NMC-204 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIA PaCa-2 cells was competed by the NMC-P2 peptide (B), and the binding of monoclonal antibody NMC-303 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIA PaCa-2 cells was competed by the NMC-P3 peptide (C).

FIG. 9 shows that the binding of monoclonal antibody NMC-103 to its extracellularly accessible epitope of HDM2 on the plasma membrane of intact human pancreatic cancer MIA PaCa-2 cells was competed by the full-length recombinant HDM2 protein.

FIG. 10 shows the cell-ELISA binding results of monoclonal antibody NMC-103, monoclonal antibody NMC-204, an antibody against E-cadherin, and an antibody against Cytochrome-C to intact human pancreatic MiaPaCa-2 cells.

FIGS. 11A-C present flow cytometry data on % cells stained with monoclonal antibodies NMC-103 (A), NMC-204 (B), and anti-Na+/K+ ATPase α-1 (C), respectively.

FIGS. 12A-D show that monoclonal antibodies NMC-103 (A and D) and NMC-204 (B and D), but not an anti-Cytochrome-C antibody (C and D), inhibited cell proliferation of intact human pancreatic MIAPaCa-2 cells.

FIGS. 13A-C show that monoclonal antibody NMC-103 (B and C) in the presence of normal human serum induces complement-mediated cytotoxicity against human pancreatic MIAPaCa-2 cells as compared with cells treated with normal human serum in the absence of any antibody (A).

FIGS. 14A-B show the lack of binding of many commercially available monoclonal antibodies to either NMC-P1 (A) or NMC-P2 (B).

FIGS. 15A-B show that an anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) reacted with intact cancer cells (A), but an anti-HDM2 antibody termed “Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)” (EMD Millipore, Cat. No. OP144) and an anti-HDM2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” (EMD Millipore, Cat. No. OP46) did not react with intact cancer cells (B).

FIG. 16 depicts the effect of monoclonal antibody NMC-204 on tumor volume of the LL/2 syngeneic mouse model of lung cancer.

FIGS. 17A-B depict the effect of monoclonal antibody NMC-103 on tumor volume (A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of colon cancer.

FIGS. 18A-B depict the effect of monoclonal antibody NMC-204 on tumor volume (A) and tumor cell proliferation (B) of the MC-38 syngeneic mouse model of colon cancer.

FIG. 19 depicts the effect of monoclonal antibody NMC-103 alone (2 mg/kg), a combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), a combination of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103 (2 mg/kg), and isotype control mouse IgG1 (2 mg/kg), respectively, on tumor volume of the Panc-2 syngeneic mouse model of pancreatic cancer. Treatment started when tumors in mice reached approximately 70 mm³.

FIG. 20 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-103.

FIG. 21 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-204.

FIG. 22 depicts the DNA sequence and protein sequence of the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody NMC-303. The leader sequence before the DNA and protein sequences of the heavy chain variable region and the light chain variable region is in bold (but not underlined).

FIG. 23 shows the tumor size of mice treated with the anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01), the tumor size of mice treated with NMC-103, the tumor size of mice treated with NMC-204, and the tumor size of mice treated with isotype control.

FIGS. 24A-F. Anti-HDM2-specific antibodies stain the surface of cancer cells but not normal cells. Intact cells released either with either EDTA or Trypsin were blocked with 5% human serum albumin. Cells were then incubated with either polyclonal N-20 M(H)DM2-specific antibody (sc-813, N-20, rabbit IgG; from Santa Cruz; “N-20”) or monoclonal M(H)DM2-specific OP145 antibody (OP145, mouse IgG1; from Calbiochem; “OP145”) for 90 min. on ice. Another set of cells prepared under the same conditions were incubated with the same antibodies that were pre-incubated with their corresponding blocking peptides before incubation with cells. Following primary antibody incubation, cells were washed 3 times with ice-cold PBS followed by FITC-secondary antibody incubation for 60 min. Cells were then washed 3 times with PBS and were subjected to FACS analyzer. Human melanoma cells (24A, 24B, and 24C), primary human ovarian cancer cells (24D and 24E), and normal mouse splenocytes (24F). FIG. 24A: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. FIG. 24B: area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody. FIG. 24C: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. FIGS. 24D & E: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. FIG. 24F: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.

FIGS. 25A-C. (A) Human pancreatic or ovarian cancer cells and normal human fibroblasts were treated with normal human serum (NETS) alone, NHS+anti-HDM2 OP145 monoclonal antibody (mouse IgG1, from Calbiochem, “OP145”) or control antibody (NHS+Cytochrome C). Extensive cell death as evident by Propidium Iodide (PI) staining was observed when cancer cells were treated with the OP145 antibody (see panels b and e) in the presence of NHS, whereas the same antibody had no effect on the viability of normal human fibroblasts (see panel g). Control antibody to Cytochrome C shows no cytotoxicity (see panel c) beyond that observed in untreated cells (see panel a). Lack of cell death is manifested by no or little PI staining in panels a, c, d, f and g. The cell death marker PI was visualized using Olympus FluoView FV1000 Confocal Laser Scanning Biological Microscope built on the Olympus IX81 Inverted Microscope. (B) Rodent pancreatic cancer cells were treated with anti-HDM2 antibodies and control cytochrome C antibody. Extensive cell death as evident by Propidium Iodide (PI) staining was observed when cells were treated with anti-HDM2 antibodies, N-20 (polyclonal, sc-813 N-20, rabbit IgG, from Santa Cruz, “N-20”) or C-18 (polyclonal, sc-812 C-18, rabbit IgG; from Santa Cruz; “C-18”) (see panels b and e), whereas, no cytotoxicity was observed when cells were treated with anti-HDM2 monoclonal OP46 antibody (OP46 (Ab-1); mouse IgG1; from Calbiochem; “OP46”) (see panel d) or control Cytochrome C antibody (see panel e). (C) M(H)DM2-specific antibodies are cytotoxic to pancreatic cancer MiaPaCa-2 cells in the presence of NHS. Quantitative representations of M(H)DM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells. Cells treated with anti-M(H)DM2 (C-18) antibody in the presence of NHS demonstrated cytotoxicity over 15-30 min. post-treatment, whereas anti-HDM2 OP46 shows no cytotoxic effect beyond that observed when cells were treated with control anti-Cytochrome C antibody or when cells were treated with NHS in the absence of anti-M(H)DM2 antibodies.

FIG. 26 shows the tumor size of mice treated with anti-HDM2 antibody OP145 and the tumor size of mice treated with PBS control (in Panc02 syngeneic mouse model of pancreatic cancer). The x axis shows days after tumor cell injection into the mice. The y axis shows tumor volume in mm³. The arrow shows the day on which the treatment was started.

FIG. 27 depicts the effect of monoclonal antibody NMC-103 alone (10 mg/kg), a combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), a combination of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103 (10 mg/kg), and isotype control mouse IgG1 (10 mg/kg), respectively, on tumor volume of the Panc-2 syngeneic mouse model of pancreatic cancer (number of mice/group=8). Treatment in this study started when tumors in mice reached approximately 80-100 mm³.

FIG. 28 depicts a Kaplan Meier survival analysis demonstrating survival benefit in mice that received NMC-103 alone or in combination with chemotherapy when compared to chemotherapy alone or control antibody under the experimental conditions described in FIG. 27.

FIG. 29 shows that mice previously treated with NMC-103 as described in FIG. 27, become immune to tumor re-challenging after drug withdrawal. To evaluate the long-term anti-tumor effect of NMC-103, at 62 days after the start of the study, mice that had previously received with a combination of G+nP, mice in group C (mice that had been previously treated with NMC-103) and group D (mice that had been previously treated with a combination of NMC-103+G+nP) were re-challenged by a second round of Panc-2 inoculation (subcutaneous injection of 2×10⁶ cells/mouse), on the left dorsal flank. Tumor growth was monitored for 10 days at which point, a tumor of 90 mm³ was measured in the mice from group B. No tumor was observed in mice from the two groups that had previously received NMC-103 antibody (Groups C and D).

FIG. 30 depicts the effect of a single dose of monoclonal antibody NMC-103 (10 mg/kg) or isotype control mouse IgG1 (10 mg/kg), when added to a treatment regimen of a combination of low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg), in the treatment of large size tumors (i.e. advanced cancers) on tumor volume of the Panc-2 syngeneic mouse model of pancreatic cancer. Mice were treated with pancreatic cancer standard of care (Gemcitabine (25 mg/kg)+nab-Paclitaxel (5 mg/kg)) for 19 days at which point they reached a tumor size of approximately 450 mm³. Mice were then randomly divided in 2 groups that received a single dose of an isotype control mouse IgG1 (10 mg/kg) or NMC-103 (10 mg/kg). As shown in this figure, a single i.p. injection of NMC-103 reduced the tumor size by almost half 6 days post treatment (from 438 mm³ to 233 mm³).

FIG. 31 depicts the effect of monoclonal antibody NMC-103 on tumor volume of the MC-38 syngeneic mouse model of colon cancer. As shown in this figure, mice treated with NMC-103 at 10 mg/kg, 2 times per week for 2 weeks reached an average tumor size of 210 mm³, while mice in the group that were treated with isotype control antibody at 10 mg/kg grew rapidly and reached 1168 mm³ by day 12. When compared to mice treated with NMC-103 at 0.4 mg/kg (FIG. 17), these data support the dose-dependent anti-tumor effect of NMC-103 antibody.

FIGS. 32A and 32B depict the effect of a chimeric version of monoclonal antibody NMC-303. Isotype class-switching was performed on a mouse NMC-303 to convert it from a mouse IgM to a chimeric IgG1. The mouse Heavy and Light chain variable regions were cloned into a human Ig gamma-1 chain and human Ig kappa chain as constant region. A total of eight (8) BALB/c mice were injected subcutaneously with CT-26. Mice were then divided into two groups (n=4) that received: A) control antibody (10 mg/kg) or B) chimeric version of NMC-303 antibody (10 mg/kg) two times a week for 3 weeks. FIG. 32A shows that by day 24 post tumor inoculation, mice treated with chimeric version of NMC-303 (10 mg/kg) reached an average tumor size of 726 mm3, while mice treated with control antibody (10 mg/kg) had an average tumor size of 1746 mm3. Furthermore, FIG. 32B shows the individual mouse tumor sizes on day 24 post tumor inoculation.

FIG. 33 shows the cell-based-ELISA results of the binding of humanized monoclonal NMC-H103 (VH4/VK3) and chimeric monoclonal (NMC-C303) antibodies to membrane-bound MDM2 on intact mouse Lewis Lung (LL/2) cancer cells. An antibody to plasma membrane marker E-Cadherin also binds to intact un-permeabilized mouse LL/2 cancer cells, while antibodies to cytoplasmic proteins cytochrome C, cyclin D1 and Bcl-2 do not bind to intact un-permeabilized cells. All primary antibodies were used at 5 μg/mL. Anti-mouse and anti-human secondary HRP conjugated antibodies were used at 1:2000 to 1:4000 range.

FIGS. 34A-B show the cell-based ELISA binding results demonstrating the binding of humanized NMC-H103 (VH4/VK3) and chimeric (NMC-C303) antibodies to a panel of intact, un-permeabilized human (34A) and murine (34B) cancer cells. No binding beyond background staining was shown using anti-human secondary antibody.

FIGS. 35A-B. FIG. 35A shows the cell-based ELISA results demonstrating the binding of humanized monoclonal antibody NMC-H103 (VH4/VK3) to intact, un-permeabilized mouse LL/2 cancer cells, while pre-incubation of humanized monoclonal antibody NMC-H103 (VH4/VK3) (5 μg/mL) with NMC-P1, reduces the binding efficiency of humanized monoclonal antibody NMC-H103 (VH4/VK3) to its target. Pre-incubation of chimeric monoclonal antibody NMC-C303 with NMC-P1 has no effect on its binding to mouse LL/2 cancer cells. FIG. 35B shows flow cytometry results on live (7AAD negative) cells showing the binding of humanized monoclonal antibody NMC-H103 (VH4/VK3) (left panel; solid black curve) and chimeric monoclonal antibody NMC-C303 (right panel; solid black curve) to live OVCAR-3 (upper panel) and mouse LL/2 cancer cells (lower panel). Dashed line curves show the reduced binding of humanized monoclonal NMC-H103 (VH4/VK3) and chimeric monoclonal NMC-C303 antibodies to OVCAR-3 and mouse LL/2 cancer cells, when cells are pre-treated with trypsin.

FIGS. 36A-B show results from in vivo experiments utilizing NOD scid gamma mice (NSG: NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ) immune-deficient mice demonstrating that the anti-cancer activity of the humanized monoclonal NMC-H103 (VH4/VK3) and the chimeric monoclonal NMC-C303 antibodies is significantly more effective in presence of normal human peripheral blood monocytes (Hu-PBMCs) than in the absence of Hu-PBMCs. In FIG. 36A, mouse colon cancer MC-38 cells subcutaneously inoculated in NSG mice (n=12) and allowed to reach a tumor volume of 400 mm³ at which point they were randomly divided into 3 groups (n=4 mouse/group). Hu-PBMCs (7×10⁶ Hu-PBMCs/mouse) were injected in tail vein of mice in group 1 (filled triangle) and group 3 (filled circle), whereas mice in group 2 (filled square) did not receive any Hu-PBMCs. A week later (tumor volume of 600 mm³), mice in group 1 received intraperitoneal (i.p.) injection of isotype control antibody (10 mg/kg), while mice in groups 2 and 3 received i.p. injection of chimeric monoclonal NMC-C303 (10 mg/kg). In FIG. 36B, human lung cancer A549 cells were subcutaneously injected into NSG mice (n=12) and allowed to reach a tumor volume of 300 mm³, at which time mice were randomly divided into 3 groups (n=4 per group). Hu-PBMCs (7×10⁶ Hu-PBMCs/mouse) were injected in tail vein of mice in group 1 (left bar) & 3 (right bar), whereas mice in group 2 (middle bar) did not receive any Hu-PBMCs. Mice in group 1 received a single i.p. injection of isotype control antibody (10 mg/kg), while mice in groups 2 and 3 received a single i.p. injection of humanized monoclonal antibody NMC-H103 (VH4/VK3) (10 mg/kg). Subcutaneous tumors were measured 4 days post antibody treatment.

FIGS. 37A-C show in vitro cell proliferation MTT assay results of M109 cells treated with anti-PD1 antibody (A), results of the in vivo effect of anti-PD1 antibody on the growth of M109 tumor in NSG mice (B), and flow cytometry results on freshly excised tumors from mice treated with isotype control or anti-PD1 antibodies. FIG. 37A shows MTT cell proliferation assay results of M109 cells treated with 75 μg/mL (middle bar) or 100 μg/mL (right bar) of anti-PD1 antibody for 48 hours. As control, M109 cell proliferation was also measured in presence of isotype control antibody at 100 μg/mL (left bar). FIG. 37B depicts the effect of anti-PD1 antibody on the growth of M109 tumor in mice. NSG mice (n=8) were subcutaneously inoculated with M109 cells (50,000 cells/mouse). Two weeks later, mice were randomly divided in two groups. Mice in group 1 (solid curve) received isotype control antibody (12.5 mg/kg; xl/week), while mice in group 2 (dashed curve) received anti-PD1 antibody (12.5 mg/kg; xl/week). In FIG. 37C, tumors from mice in groups 1 and 2 (n=4) were assessed for the expression of plasma membrane-bound M(H)DM2 by flow cytometry with humanized monoclonal antibody NMC-H103 (VH4/VK3). Binding of humanized monoclonal antibody NMC-H103 (VH4/VK3) to cells of tumors from mice in group 1 (left curve) and group 2 (right curve) is shown.

FIGS. 38A-B show single cycle (A) and multiple cycle (B) sensorgrams data and fitted curves (1:1 binding model) for binding to NMC-P1 of chimeric monoclonal antibody NMC-C103 (VH0/VK0) and humanized monoclonal antibody NMC-H103 variants having VH/and VK combinations identified in the figure. The sequences of the respective VH and VK of humanized monoclonal antibody NMC-H103 variants are provided in Tables 15-27 and Section 11.

7. DETAILED DESCRIPTION

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2). In a specific embodiment, the extracellularly accessible epitope is contained within SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Antibodies provided herein are described in Section 5.1, below. Also provided herein are antibody-drug conjugates comprising an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) described herein bound (e.g., covalently) to a cytotoxic drug. Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), wherein said antibodies or fragments are not bound to a cytotoxic component.

Also provided herein are pharmaceutical compositions comprising an antibody or fragment that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) described herein. In certain embodiments, such pharmaceutical compositions comprise a therapeutically effective amount of such antibody or fragment (i.e., an amount that can be used to treat a cancer in a subject, e.g. by achieving one or more anti-tumor effects described herein).

Also provided herein are nucleic acids encoding the antibodies and antigen-binding fragments described herein. In certain embodiments, provided herein are vectors and cells comprising nucleic acids encoding such antibodies or antigen-binding fragments thereof. Cells recombinantly producing the antibodies or antigen-binding fragments thereof described herein are also provided.

Chimeric antigen receptors (CARs) are engineered receptors that provide both antigen binding and immune cell activation functions (Sadelain et al., 2013, Cancer Discovery 3:388-398). Also provided herein are CARs comprising a single-chain variable fragment (scFv) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, such as a scFv comprising the VH and VL of an anti-M(H)DM2/4 antibody described herein, fused via a linker to a transmembrane domain (e.g., of CD3 zeta) fused to an intracellular T cell activation domain such as CD3 zeta intracellular domain, optionally further fused to a co-stimulatory domain (e.g., CD28 intracellular domain). T cells expressing such CARs are also provided.

Also provided herein is a peptide, the amino acid sequence of which is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). The peptide can be, for example, synthetic or recombinant. In some embodiments, the peptide is purified. In some embodiments, the peptide is labeled with a detectable marker (e.g., a fluorescent marker or an isotope). In some embodiments, the peptide is tagged (e.g., with a GST, His, Strep, myc, FLAG, or HA tag). In some embodiments, a cysteine is added at one of the ends of the peptide (which may allow for linkage to a carrier protein). In some embodiments, the peptide is linked to a carrier protein (e.g., linked to Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, thyroglobulin, tetanus toxoid, or diphtheria toxoid). Also provided herein are nucleic acids encoding a peptide described herein. Also provided herein are vectors and cells comprising a nucleic acid encoding a peptide described herein. Cells recombinantly producing a peptide described herein are also provided. Also provided herein are uses of the peptides described herein as immunogens. The peptides of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 described herein contain extracellularly accessible epitopes of MDM2 and HDM2. In particular, provided herein are methods of making an anti-M(H)DM2/4 antibody (e.g., an antibody that specifically binds to M(H)DM2/4) by immunizing an animal (e.g., a mouse or a rabbit) with a peptide described herein. Also provided herein are methods of screening antibodies for binding to one or more of the peptides described herein (for example, using ELISA with a plate-bound peptide). Also provided herein are methods of identifying an anti-M(H)DM2/4 antibody suitable for therapeutic use in treating cancer or preventing metastasis, or suitable for use in diagnosis of cancer, by contacting an anti-M(H)DM2/4 antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide, and detecting or measuring binding between the antibody and the peptide that occurs, where the detection of binding between the antibody and the peptide indicates that the antibody is suitable for the therapeutic or diagnostic use. For example, provided herein are methods of identifying an anti-M(H)DM2/4 antibody suitable for therapeutic use by contacting an anti-M(H)DM2/4 antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide, and detecting or measuring binding between the antibody and the peptide that occurs, and, if the binding between the antibody and the peptide is detected, using the antibody in the methods of treating cancer described herein. In another example, provided herein are methods of identifying an anti-M(H)DM2/4 antibody suitable for diagnostic use by contacting an anti-M(H)DM2/4 antibody with a peptide described herein under conditions suitable for binding between the antibody and the peptide, and detecting or measuring binding between the antibody and the peptide that occurs, and, if the binding between the antibody and the peptide is detected, using the antibody in the methods of diagnosing cancer described herein.

Also provided herein are methods for treating cancer, inhibiting tumor growth or proliferation, inhibiting tumor progression, and/or preventing metastases in a subject by administering to the subject an anti-M(H)DM2/4 antibody or fragment described herein, in particular, an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (in particular, a region exposed on the plasma membrane surface of cancer cells). Preferably the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2 (in particular, a region exposed on the plasma membrane surface of cancer cells). In certain embodiments, the antibody or fragment (e.g., for use in the methods described herein) does not bind or only minimally binds to the plasma membrane surface of normal cells of the tissue type from which the cancer in the subject originates. In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment is any chimeric or humanized anti-M(H)DM2 antibody described herein.

Also provided herein are methods for treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration of an inhibitor of one or more inhibitory checkpoint molecules by administering to the subject an anti-M(H)DM2/4 antibody or fragment described herein, in particular, an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 (in particular, a region exposed on the plasma membrane surface of cancer cells). Preferably the antibody or fragment thereof specifically binds to an extracellularly accessible epitope of HDM2 (in particular, a region exposed on the plasma membrane surface of cancer cells). In certain embodiments, the antibody or fragment (e.g., for use in the methods described herein) does not bind or only minimally binds to the plasma membrane surface of normal cells of the tissue type from which the cancer in the subject originates. In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment is any chimeric or humanized anti-M(H)DM2 antibody described herein.

Where the subject being treated is a human, in certain embodiments, an antibody or a fragment thereof used herein specifically binds to an extracellularly accessible epitope of HDM2 and/or HDM4 (a region exposed on the plasma membrane surface of cancer cells). In one embodiment of treating a human, an antibody or a fragment thereof used herein specifically binds to an extracellularly accessible epitope of HDM2 (a region exposed on the plasma membrane surface of cancer cells) (optionally, such antibody or fragment that does not bind to HDM4). In one embodiment of treating a human, an antibody or a fragment thereof used herein specifically binds to an extracellularly accessible epitope of HDM4 (a region exposed on the plasma membrane surface of cancer cells) (optionally, such antibody or fragment that does not bind to HDM2).

Where the subject is a non-human animal (e.g., a mammal such as a dog or a cat), an antibody or a fragment thereof used herein binds to an extracellularly accessible epitope of M(H)DM2/4 (a region exposed on the plasma membrane surface of cancer cells), where the M(H)DM2/4 is a homologue of HDM2 and/or HDM4 expressed in such animal. In one embodiment, an antibody or a fragment thereof used herein binds to an extracellularly accessible epitope of M(H)DM2 (a region exposed on the plasma membrane surface of cancer cells), where the M(H)DM2 is a homologue of HDM2 expressed in such animal (optionally, such antibody or fragment does not bind to M(H)DM4). In one embodiment, an antibody or a fragment thereof used herein binds to an extracellularly accessible of M(H)DM4 (a region exposed on the plasma membrane surface of cancer cells), where the M(H)DM4 is a homologue of HDM4 expressed in such animal (optionally, such antibody or fragment does not bind to M(H)DM2).

The description of the invention that follows is largely in terms of HDM2 and antibodies and antibody fragments thereto, which shall be understood to be for use in treating a human; it will be clear to one skilled in the art that the description also should be deemed applicable to: (i) HDM4 and antibodies and antibody fragments thereto, and use thereof for treatment of humans (unless indicated otherwise explicitly or by context), and (ii) M(H)DM2/4 and antibodies and antibody fragments thereto, and use thereof for treatment of non-human animals, e.g., mammals (unless indicated otherwise explicitly or by context). In preferred embodiments, the patients or subjects being treated using the methods described herein are human.

In preferred embodiments, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein mediates complement-dependent cytotoxicity (CDC), mediates antibody-dependent cell-mediated cytotoxicity (ADCC), and/or is bound to a cytotoxic drug or drugs (e.g., is an antibody-drug conjugate). In preferred embodiments, the invention provides for the use of antibodies that mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

In certain embodiments, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not bound to a cell-penetrating peptide. Cell penetrating peptides can insert into a cell plasma membrane and transport molecules to which they are attached into the cell. Such cell-penetrating peptides include, without limitation, a membrane resident peptide (MRP), Membrane Transduction Domain of Antennapedia, trans-activating transcriptional activator (TAT), and a Penetratin peptide. In certain embodiments, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to a membrane resident peptide (MRP), Membrane Transduction Domain of Antennapedia, TAT, and/or a Penetratin peptide. In certain embodiments, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to any peptide sequence that can insert into the lipid bilayer of the plasma membrane of cells. In one embodiment, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to an MRP. In one embodiment, the anti-HDM2 antibody or a fragment thereof used in accordance with the methods described herein is not attached to a Penetratin peptide.

The examples set forth herein demonstrate that HDM-2 targeting antibodies alone are selectively cytotoxic to cancer cells. Further, as set forth in the examples herein, it has been demonstrated that extracellularly accessible epitopes of HDM2 are appropriate therapeutic targets for anti-HDM2 antibodies, and that cancer cells expressing HDM2 on their surface can be successfully targeted and destroyed with antibodies to such extracellular regions of HDM2. In particular, the data presented in the examples demonstrate that select HDM2-specific antibodies can bind to the extracellularly accessible sequences of HDM2 on the surface membrane of intact cancer cells, while exhibiting minimal binding to the surface membrane of normal human blood mononuclear cells. In addition, the data presented in the examples show that such HDM2-specific antibodies can inhibit the growth of cancer cells in vitro and in vivo, strongly suggesting that they can be used as therapeutic agents in vivo. Further, the data presented in the examples show that such HDM2-specific antibodies can have a synergistic anti-tumor effect when combined with chemotherapeutic drugs. In addition, the data presented in the examples show that certain chimeric and humanized HDM2-specific antibodies described herein have anti-tumor effect. Further, the data presented in the examples show that subjects whose tumors undergo hyper-progression in response to treatment with an inhibitor of an inhibitory immune checkpoint molecule have an increased level of plasma-membrane-bound M(H)DM2 that could be targeted with HDM2-specific antibodies that bind to the extracellularly accessible epitopes of M(H)DM2 (e.g., certain chimeric and humanized HDM2-specific antibodies described herein).

7.1 Antibodies

Provided herein are antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) (a region exposed on the plasma membrane surface of cells). “Specifically bind[s]/binding” as those terms are used herein does not exclude cross-reactivity of the antibody or antigen-binding fragment; thus, for example, antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of HDM2 exposed on the plasma membrane surface of cells may also specifically bind to (cross-react with) MDM2. In particular, provided herein are anti-M(H)DM2/4 antibodies and fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 and that have an anti-tumor effect (e.g., inhibit tumor growth in vivo). In specific embodiments, an antibody or an antigen-binding fragment thereof specifically binds an epitope of M(H)DM2/4 that is extracellularly accessible on cancer cells but not on non-cancer cells (e.g., non-cancerous cells of the same organ type or tissue type as the cancer cells). In other specific embodiments, an antibody or an antigen-binding fragment thereof specifically binds an epitope of M(H)DM2/4, exposure or accessibility of which on the plasma membrane surface of cancer cells is increased relative to its exposure or accessibility on the plasma membrane surface of non-cancer cells (e.g., non-cancerous cells of the organ or tissues of the host). Also provided herein are antibodies or antigen-binding fragments thereof that (immuno) specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2), which are not bound to a cell-penetrating peptide (e.g., a membrane resident peptide).

Also provided herein are antibodies or antigen-binding fragments thereof that (immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an extracellularly accessible epitope of M(H)DM2/4, wherein the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that (immuno) specifically bind to M(H)DM2/4 (e.g., HDM2), in particular to an extracellularly accessible epitope of M(H)DM2/4 wherein the antibody or fragment specifically binds to a peptide the sequence of which peptide consists of MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3); and wherein such antibodies or fragments have an anti-tumor effect in vivo, and/or wherein such antibodies or fragments are not bound to a cell-penetrating peptide.

Also provided herein are anti-M(H)DM2/4 antibodies and fragments having heavy chain variable regions and/or light chain variable regions described herein (see, e.g., having sequences of heavy chain variable regions and/or light chain variable regions of antibodies NMC-103, NMC-204 and NMC-303 provided herein, see, e.g., Section 8 and FIGS. 20-22). Also provided herein are anti-M(H)DM2/4 antibodies and fragments having one or more complementarity determining regions (CDRs) described herein (see, e.g., CDRs provided in Tables 4-9 and FIGS. 20-22).

Also provided herein are chimeric and humanized anti-M(H)DM2/4 antibodies and fragments having heavy chain variable regions and/or light chain variable regions described herein (e.g., having sequences of heavy chain variable regions and/or light chain variable regions of antibodies NMC-C303, NMC-C103 and variants of NMC-H103 provided herein, see, e.g., Sections 9 and 11, and Tables 11-27). Also provided herein are anti-M(H)DM2/4 antibodies and fragments having one or more complementarity determining regions (CDRs) described herein (see, e.g., CDRs provided in Tables 11-27) (e.g., chimeric or humanized antibodies). Also provided herein are chimeric and humanized anti-M(H)DM2/4 antibodies and fragments having one or more framework regions (FR) described herein (see, e.g., heavy chain framework regions (HFR) and light chain framework regions (LFR) provided in Tables 11-27).

CDRs are defined in various ways in the art, including the Kabat, Chothia, AbM, Contact, and IMGT. In certain aspects, the CDRs of an antibody can be defined according to the Kabat system, which is based on sequence variability (see, e.g., Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391; Kabat E A et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. In a specific embodiment, with respect to the Kabat system, (i) the VH CDR1 is present at amino acid positions 31 to 35 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 50 to 68 or 50 to 66 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain. In a specific embodiment, with respect to the Kabat system, (i) the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain; (ii) the VH CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain; and (iii) the VH CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain. As is well known to those of skill in the art, with respect to the Kabat system, the actual linear amino acid sequence of the antibody variable domain can contain fewer or additional amino acids due to a shortening or lengthening of a framework region (FR) and/or CDR and, as such, an amino acid's Kabat number is not necessarily the same as its linear amino acid number. The Kabat CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the Kabat system.

In certain aspects, the CDRs of an antibody can be defined according to the Chothia system, which is based on the location of immunoglobulin structural loop regions (see, e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No. 7,709,226). The term “Chothia CDRs,” and like terms are recognized in the art and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, which will be referred to herein as the “Chothia CDRs” (see also, e.g., U.S. Pat. No. 7,709,226 and Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). In a specific embodiment, with respect to the Chothia system, using the Kabat numbering system of numbering amino acid residues in the VH region, (i) the VH CDR1 is present at amino acid positions 26 to 32 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 52 to 59 or 52 to 57 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain. In a specific embodiment, with respect to the Chothia system, using the Kabat numbering system of numbering amino acid residues in the VL region, (i) the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain; (ii) the VL CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain; and (iii) the VL CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain. The Chothia CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the Chothia system.

In certain aspects, the CDRs of an antibody can be defined according to the AbM system, which is based on AbM hypervariable regions that represent a compromise between the Kabat CDRs and Chothia structural loops, and where CDRs are determined using Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, with respect to the AbM system, using the Kabat numbering system of numbering amino acid residues in the VH region, (i) the VH CDR1 is present at amino acid positions 26 to 35 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 50 to 61 or 50 to 59 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 101 to 105 or 99 to 104 or 99 to 106 of the heavy chain. In a specific embodiment, with respect to the AbM system, using the Kabat numbering system of numbering amino acid residues in the VL region, (i) the VL CDR1 is present at amino acid positions 24 to 39 or 24 to 34 of the light chain; (ii) the VH CDR2 is present at amino acid positions 55 to 61 or 50 to 56 of the light chain; and (iii) the VH CDR3 is present at amino acid positions 94 to 102 or 89 to 97 of the light chain. The AbM CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the AbM numbering system.

In certain aspects, the CDRs of an antibody can be defined according to the IMGT system (see “IMGT®, the international ImMunoGeneTics information system® website imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entirety). In a specific embodiment, with respect to the IMGT system, (i) the VH CDR1 is present at amino acid positions 27 to 33 or 26 to 33 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 51 to 60 or 51 to 58 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 99 to 105 or 97 to 103 of the heavy chain. In a specific embodiment, with respect to the IMGT system, (i) the VL CDR1 is present at amino acid positions 27 to 37 of the light chain; (ii) the VH CDR2 is present at amino acid positions 55 to 57 of the light chain; and (iii) the VH CDR3 is present at amino acid positions 94 to 102 of the light chain. The IMGT CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the IMGT system.

In certain aspects, the CDRs of an antibody can be defined according to the Contact system. The Contact definition is based on an analysis of the available complex crystal structures (bioinf.org.uk/abs) (see MacCallum R M et al., (1996) J Mol Biol 5: 732-745; see also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). In a specific embodiment, with respect to the Contact system, using the Kabat numbering system of numbering amino acid residues in the VH region, (i) the VH CDR1 is present at amino acid positions 30 to 35 of the heavy chain; (ii) the VH CDR2 is present at amino acid positions 47 to 61 or 47 to 59 of the heavy chain; and (iii) the VH CDR3 is present at amino acid positions 99 to 104 or 97 to 103 or 97 to 105 of the heavy chain. In a specific embodiment, with respect to the Contact system, using the Kabat numbering system of numbering amino acid residues in the VL region, (i) the VL CDR1 is present at amino acid positions 30 to 41 or 30 to 36 of the light chain; (ii) the VH CDR2 is present at amino acid positions 51 to 60 or 46 to 55 of the light chain; and (iii) the VH CDR3 is present at amino acid positions 94 to 101 or 89 to 96 of the light chain. The Contact CDR positions may vary depending on the antibody, and may be determined according to methods known in the art. In a specific embodiment, the CDRs of the antibodies described herein are determined using the Contact system.

In a particular embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise CDRs of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303), which are defined according to any of the above-described systems.

In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs (preferably all three VH CDRs) of any anti-HDM2 antibody described herein (such as NMC-103, NMC-204, or NMC-303). As is known in the art, VHs contain VH CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4), optionally the framework regions are human framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:36 (which is the VH of NMC-103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:38 (which is the VH of NMC-204). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-303). In certain embodiments, such antibody or fragment is a humanized antibody or fragment.

In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs (preferably all three VL CDRs) of any anti-HDM2 antibody described herein (such as NMC-103, NMC-204, or NMC-303). As is known in the art, VLs contain VL CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4); optionally the framework regions are human framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:37 (which is the VL of NMC-103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:39 (which is the VL of NMC-204). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:40 (which is the VL of NMC-303). In certain embodiments, such antibody or fragment is a humanized antibody or fragment.

In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs (preferably all three VH CDRs) of any anti-HDM2 antibody described herein (such as NMC-103, NMC-204, or NMC-303) and comprise a light chain variable region (VL) having one, two or all three VL CDRs (preferably all three VL CDRs) of such anti-HDM2 antibody.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 4 (providing VH CDRs of NMC-103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 4. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 5 (providing VL CDRs of NMC-103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 4; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 5.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 6 (providing VH CDRs of NMC-204). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 6. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 7 (providing VL CDRs of NMC-204). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 6; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 7.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 8 (providing VH CDRs of NMC-303). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 8. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 9 (providing VL CDRs of NMC-303). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 8; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 9.

In certain embodiments described herein referencing an antibody or fragment thereof comprising a sequence (e.g., VH or VL sequence) or sequences having a certain percent identity (which is less than 100%) to a sequence, the antibody or fragment thereof comprises the sequence (e.g., VH or VL sequence) and not the sequences having a certain percent identity (which is less than 100%) to the sequence. In certain embodiments, described herein referencing an antibody or fragment thereof comprising sequences having a certain percent identity (which is less than 100%) to a sequence, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH of any antibody described herein, such as a VH of any antibody provided in Section 8 or FIGS. 20-22 (e.g., the VH of NMC-103, the VH of NMC-204, or the VH of NMC-303), or a VH having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VL of any antibody described herein, such as a VL of any antibody provided in Section 8 or FIGS. 20-22 (e.g., the VL of NMC-103, the VL of NMC-204, or the VL of NMC-303), or a VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH and a VL of any antibody described herein, such as a VH and VL of any antibody provided in Section 8 or FIGS. 20-22 (e.g., the VH and VL of NMC-103, the VH and VL of NMC-204, or the VH and VL of NMC-303), or a VH and VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:36, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:37, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:38, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:39, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:41, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto.

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Kabat VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more Kabat VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Kabat VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Kabat VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three Kabat VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Chothia VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more Chothia VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Chothia VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Chothia VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three Chothia VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more AbM VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more AbM VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise AbM VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three AbM VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three AbM VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Contact VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more Contact VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise Contact VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Contact VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three Contact VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more IMGT VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or one or more IMGT VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise IMGT VH CDR 3 of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three IMGT VL CDRs of a VL of any one of the antibodies described herein (any one of NMC-103, NMC-204, and NMC-303) and/or three IMGT VH CDRs of a VH of any one of the antibodies described herein (any one of antibodies NMC-103, NMC-204, and NMC-303).

In a particular embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise CDRs of any one of the antibodies described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103), which are defined according to any of the above-described systems.

In certain embodiments, provided herein are antibodies (e.g., chimeric or humanized antibodies) or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs (preferably all three VH CDRs) of any anti-HDM2 antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in the art, VHs contain VH CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4), optionally the framework regions are humanized or human framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-C303). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:283 (which is the VH of NMC-C103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:287 (which is the VH1 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:291 (which is the VH2 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:295 (which is the VH3 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:299 (which is the VH4 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:305 (which is the VH6 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs of a VH having the amino acid sequence of SEQ ID NO:309 (which is the VH7 variant of NMC-H103).

In certain embodiments, provided herein are chimeric or humanized antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs (preferably all four VH FRs) of any anti-HDM2 antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in the art, VHs contain VH CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VH: FR1-VH CDR 1-FR2-VH CDR 2-FR3-VH CDR 3-FR4), optionally the framework regions are humanized or human framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:40 (which is the VH of NMC-C303). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:283 (which is the VH of NMC-C103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:287 (which is the VH1 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:291 (which is the VH2 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:295 (which is the VH3 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:299 (which is the VH4 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:305 (which is the VH6 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two, three or all four VH FRs of a VH having the amino acid sequence of SEQ ID NO:309 (which is the VH7 variant of NMC-H103).

In certain embodiments, provided herein are antibodies (e.g., chimeric or humanized antibodies) or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs (preferably all three VL CDRs) of any anti-HDM2 antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in the art, VLs contain VL CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4); optionally the framework regions are human or humanized framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:41 (which is the VL of NMC-C303). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:285 (which is the VL of NMC-C103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:289 (which is the VK1 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:293 (which is the VK2 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:297 (which is the VK3 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:301 (which is the VK4 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:303 (which is the VK5 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:307 (which is the VK6 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two or all three VL CDRs of a VL having the amino acid sequence of SEQ ID NO:311 (which is the VK7 variant of NMC-H103).

In certain embodiments, provided herein are chimeric or humanized antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs (preferably all four VL FRs) of any anti-HDM2 antibody described herein (such as NMC-C303, NMC-C103 and variants of NMC-H103). As is known in the art, VLs contain VL CDRs surrounded by framework regions (the CDR and FR sequences appear in the following sequence in the VL: FR1-VL CDR 1-FR2-VL CDR 2-FR3-VL CDR 3-FR4), optionally the framework regions are humanized or human framework regions. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:41 (which is the VL of NMC-C303). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:285 (which is the VL of NMC-C103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:289 (which is the VL1 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:293 (which is the VL2 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:297 (which is the VL3 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:301 (which is the VL4 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:303 (which is the VL5 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:307 (which is the VL6 variant of NMC-H103). In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a light chain variable region (VL) having one, two, three or all four VL FRs of a VL having the amino acid sequence of SEQ ID NO:311 (which is the VL7 variant of NMC-H103).

In certain embodiments, provided herein are chimeric or humanized antibodies or antigen-binding fragments thereof that specifically bind to an extracellularly accessible epitope of M(H)DM2/4 (e.g., HDM2) and comprise a heavy chain variable region (VH) having one, two or all three VH CDRs (preferably all three VH CDRs) of any chimeric or humanized anti-HDM2 antibody described herein (such as NMC-C103, NMC-C303, or variants of NMC-H103) and comprise a light chain variable region (VL) having one, two or all three VL CDRs (preferably all three VL CDRs) of such anti-HDM2 antibody.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 11 (providing VH CDRs of NMC-C303). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 11. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 12 (providing VL CDRs of NMC-C303). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 11; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 12.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 13 (providing VH CDRs of NMC-C103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 13. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 14 (providing VL CDRs of NMC-C103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 13; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 14. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 13 (providing VH FRs of NMC-C103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 14 (providing VL FRs of NMC-C103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 13; and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 14.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 15 (providing VH1 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 15. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 16 (providing VK1 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 15; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 16.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 15 (providing VH1 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 16 (providing VK1 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 17 (providing VH2 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 17. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 18 (providing VK2 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 17; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 18.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 17 (providing VH2 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 18 (providing VK2 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 19 (providing VH3 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 19. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 20 (providing VK3 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 19; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 20.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 19 (providing VH3 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 20 (providing VK3 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 21 (providing VH4 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 21. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 22 (providing VK4 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 21; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 22.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 21 (providing VH4 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 22 (providing VK4 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 23 (providing VK5 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 23 (providing VK5 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 24 (providing VH6 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 24. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 25 (providing VK6 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 24; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 25.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 24 (providing VH6 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 25 (providing VK6 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 26 (providing VH7 variant CDRs of NMC-H103). In one embodiment, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a VH CDR3 (“CDR-H3”) identified in Table 26. In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 27 (providing VK7 variant CDRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two or all three VH CDRs identified in Table 26; and comprises a light chain variable region (VL) having one, two or all three VL CDRs identified in Table 27.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in Table 26 (providing VH7 variant FRs of NMC-H103). In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in Table 27 (providing VK7 variant CDRs of NMC-H103).

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in any one of Tables 15, 17, 19, 21, 24, and 26; and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in any one of Tables 16, 18, 20, 22, 23, 25, and 27.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three or VH CDRs identified in any one of Tables 15, 17, 19, 21, 24, and 26; and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in any one of Tables 16, 18, 20, 22, 23, 25, and 27.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in any one of Tables 15, 17, 19, and 21; and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in any one of Tables 16, 18, 20, 22, and 23.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in any one of Tables 15, 17, 19, and 21; and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in any one of Tables 16, 18, 20, 22, and 23.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, three or all four VH FRs identified in any one of Tables 21, 24, and 26; and comprises a light chain variable region (VL) having one, two, three or all four VL FRs identified in any one of Tables 20, 25, and 27.

In certain embodiments, provided herein is an antibody or a fragment thereof that specifically binds to HDM2 and comprises a heavy chain variable region (VH) having one, two, or all three VH CDRs identified in any one of Tables 21, 24, and 26; and comprises a light chain variable region (VL) having one, two, or all three VL CDRs identified in any one of Tables 20, 25, and 27.

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH of any antibody described herein, such as a VH of any antibody provided in Sections 9 and 11 (e.g., the VH of NMC-C103, the VH of NMC-C303, or the VH of any of NMC-H103 variants), or a VH having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VL of any antibody described herein, such as a VL of any antibody provided in Sections 9 and 11 (e.g., the VL of NMC-C103, the VL of NMC-C303, or the VL of any of NMC-H103 variants), or a VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising a VH and a VL of any antibody described herein, such as a VH and VL of any antibody provided in Sections 9 and 11 (e.g., the VH and VL of NMC-C103, the VH and VL of NMC-C303, or the VH and VL of any NMC-H103 variant, e.g., the VH and VL of any NMC-H103 variant tested for binding to NMC-P1 as described in Section 9.6, Example 18 and FIGS. 38A and 38B), or a VH and VL having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:41, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:40, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ ID NO:41, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:283, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:285, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:283, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ ID NO:285, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:287, 291, 295, 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:289, 293, 297, 301, 303, 307, or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:287, 291, 295, 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ ID NO:289, 293, 297, 301, 303, 307, or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:287, 291, 295, or 299, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:289, 293, 297, 301, or 303, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO:287, 291, 295, or 299, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ ID NO:289, 293, 297, 301, or 303, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO: 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and/or (ii) a VL having the amino acid sequence of SEQ ID NO:297, 307, or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, provided herein is an antibody or fragment thereof that specifically binds to HDM2 comprising: (i) a VH having the amino acid sequence of SEQ ID NO: 299, 305, or 309, or a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto; and (ii) a VL having the amino acid sequence of SEQ ID NO:297, 307, or 311, or a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity thereto. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the FRs).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Kabat VL CDRs and VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Kabat VH CDRs and VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Kabat VL CDRs and four Kabat VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or three Kabat VH CDRs and four Kabat VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Chothia VL CDRs and VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Chothia VH CDRs and VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Chothia VL CDRs and four Chothia VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or three Chothia VH CDRs and four Chothia VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more AbM VL CDRs and VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more AbM VH CDRs and VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three AbM VL CDRs and four AbM VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or three AbM VH CDRs and four AbM VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more Contact VL CDRs and VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more Contact VH CDRs and VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three Contact VL CDRs and four Contact VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or three Contact VH CDRs and four Contact VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103).

In certain aspects, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise one or more IMGT VL CDRs and VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or one or more IMGT VH CDRs and VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103). In one embodiment, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise three IMGT VL CDRs and four IMGT VL FRs of a VL of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103), and/or three IMGT VH CDRs and four IMGT VH FRs of a VH of any one of the chimeric or humanized antibodies described herein (such as NMC-C103, NMC-C303, and variants of NMC-H103).

In certain embodiments, provided herein are antibodies or fragments thereof that specifically bind to M(H)DM2/4 (e.g., HDM2) and comprise combinations of Kabat CDRs, Chothia CDRs, AbM CDRs, IMGT CDRs, and Contact CDRs (or a combination of CDRs defined by any two, three, four or five of these CDR defining systems).

In a specific embodiment, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in one embodiment, the position defining a CDR of any of antibody described herein (any one of antibodies NMC-103, NMC-204, and NMC-303) may vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to M(H)DM2/4 (e.g., HDM2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

In specific embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody described herein is a humanized immunoglobulin (e.g., an IgG) that comprises the 3 VH CDRs and the 3 VL CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) of any of the antibodies described herein (any one of murine antibodies NMC-103, NMC-204, and NMC-303), respectively, human or human-derived framework regions, and human or human-derived constant regions; antigen-binding fragments of such humanized antibodies are also provided by the present invention. Non-limiting examples of human framework regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, a humanized anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VH framework regions that are human framework regions or derived from human framework regions. In certain embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises a VL with VL CDR1, VL CDR2, and VL CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework regions that are human framework regions or derived from human framework regions. In certain embodiments, an anti-M(H)DM2/4 (e.g., HDM2) antibody or antigen-binding fragment thereof comprises (i) a VH with VH CDR1, VH CDR2, and VH CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VH framework regions that are human framework regions or derived from human framework regions; and (ii) a VL with VL CDR1, VL CDR2, and VL CDR3 as described herein (e.g., those of MNC-103, NMC-204, or NMC-303), surrounded by VL framework regions that are human framework regions or derived from human framework regions.

Human framework regions that may be used include, without limitation: (i) framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); (ii) framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); (iii) human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and (iv) framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)). See, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions.

In a specific embodiment, using routine recombinant DNA techniques, one or more of the CDRs of an anti-M(H)DM2/4 (e.g., HDM2) or antigen-binding fragment thereof described herein may be inserted within known framework regions. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions.

In certain embodiments, described herein are polynucleotides comprising combinations of the framework regions and CDRs that encode an anti-M(H)DM2/4 (e.g., HDM2) or antigen-binding fragment thereof that specifically binds M(H)DM2/4 (e.g., HDM2). One or more (e.g., one or two or three) amino acid substitutions may be made within the framework regions, preferably, one or more (e.g, one or two or three) amino acid substitutions may be made that improve binding of the antibody to M(H)DM2/4 (e.g., HDM2).

In an alternative embodiment wherein the antibody or fragment thereof is not humanized, the framework regions in the variable domains can be those of the native (e.g., murine) antibody).

Antibodies provided herein include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules that possess an antigen-binding site) that specifically bind to an extracellular region (epitope) of M(H)DM2/4 accessible on the plasma membrane surface of cancer cells (for example, an epitope that is expressed or exposed on the plasma membrane of cancer cells at greater levels than on non-cancer cells (e.g., when such cancer and non-cancer cells originated from the same tissue)).

In a preferred embodiment, anti-M(H)DM2/4 antibodies described herein are monoclonal antibodies or fragments thereof. The antibodies and fragments described herein are preferably human, humanized or chimeric. A human antibody can be a human immunoglobulin, which may be isolated from a human immunoglobulin library or isolated from mice or other animals that express antibodies from human genes. In one embodiment, an antibody provided herein is human (or a fragment of a human antibody). In one embodiment, an antibody provided herein is humanized (or a fragment of a humanized antibody). In one embodiment, an antibody provided herein is chimeric (or a fragment of a chimeric antibody) (where a chimeric antibody is an antibody with a variable region of one species (e.g., murine) and a constant region of another species (e.g., human)). Preferably, an antibody provided herein is a human, humanized or chimeric monoclonal antibody (which is particularly suitable for treatment of human subjects). In one embodiment, an antibody provided herein is a synthetic antibody. In one embodiment, an antibody provided herein is a multi-specific antibody (e.g., a bi-specific antibody). In one embodiment, an antibody provided herein is a single chain antibody, e.g., a single chain Fv (scFv). In one embodiment, an antigen-binding fragment of an anti-M(H)DM2/4 antibody is provided herein wherein the fragment can be, without limitation, an Fv fragment, a Fab fragment, a F(ab′) fragment, a F(ab′)2 fragment, or a disulfide-linked Fv (sdFv). In one embodiment, an antigen-binding fragment provided herein is an Fv fragment. In one embodiment, an antigen-binding fragment provided herein is a Fab fragment. In one embodiment, an antigen-binding fragment provided herein is a F(ab′)2 fragment. In one embodiment, an antigen-binding fragment provided herein is a F(ab′) fragment.

In a specific embodiment, an antibody provided herein is a multispecific antibody (such as a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells and specifically binds to a second antigen, wherein such binding allows re-targeting of effector cells towards tumor cells (as an example of such engineered bi-specific antibodies directed to a different target see Chames et al., 2009, MAbs 1:539-547, describing an antibody termed catumaxomab, a T-cell targeting agent). In one embodiment, an antibody provided herein is a multispecific antibody (such as a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also binds to an antigen exposed on the plasma membrane surface of an effector cell. Effector cells include but are not limited to T cells, natural killer cells, neutrophils, macrophages, dendritic cells and B lymphocytes. In one embodiment, an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of T cells (e.g., cytotoxic T cells). In one embodiment, an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to CD3. In specific embodiments, an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of natural killer cells, neutrophils, macrophages, dendritic cells, and/or B-lymphocytes. In specific embodiments, an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of neutrophils, macrophages, dendritic cells, and/or B-lymphocytes. In specific embodiments, an antibody provided herein is a multispecific antibody (e.g., a bi-specific antibody) that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 exposed on the plasma membrane surface of cancer cells, and also specifically binds to an antigen exposed on the surface of natural killer cells, macrophage and/or dendritic cells.

In a preferred embodiment, wherein a human subject is treated, the antibody used is a monoclonal antibody or an antigen-binding fragment thereof that is human, humanized or chimeric.

In certain embodiments, for example where non-human subjects are being treated, such as cats, dogs, cows, and other domestic, farm and wild animals, the antibody can be an antibody or fragment appropriate for use in the treated species (i.e., of that species). The antibodies described herein can be from any animal species, such as mammals (e.g., mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse, dog, cat) or birds (e.g., chicken).

In specific embodiments, wherein the antibody is an immunoglobulin, the immunoglobulin molecules that can be used are of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) or subclass of immunoglobulin molecule. In a preferred embodiment, the antibody is an immunoglobulin, and, in particular, an IgG. In another embodiment, the antibody is an IgM.

In preferred embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytoxicity (ADCC), and/or cytotoxicity due to a cytotoxic drug bound to the antibody or fragment.

In specific embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that are capable of inducing cytotoxicity against the cancer cells being targeted by such antibodies or fragments, where the cytotoxicity can be due to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or due to cytotoxicity of a drug bound to the antibody (where the antibody used is in a form of an antibody-drug conjugate).

In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC). Methods of making an antibody that has CDC function are known in the art. In some embodiments, in which the CDC activity is desired, the Fc region of the antibody described herein is of a human IgG (e.g., IgG1, IgG2, IgG3, IgG4) type or a human IgM type. In some embodiments, in which the CDC activity is desired, the Fc region of the antibody described herein is of a mouse IgG (e.g., IgG1, IgG2a, IgG2b, IgG3) or mouse IgM type. In one embodiment, the Fc region of the antibody described herein is of a human IgG1 isotype. In one embodiment, the Fc region of the antibody described herein is of a human IgG3 isotype. In one embodiment, the Fc region of the antibody described herein is of a human IgG2 isotype. In one embodiment, the Fc region of the antibody described herein is bioengineered (e.g., mutated) to increase its CDC activity. In one embodiment, the antibody or fragment is a bispecific antibody or fragment that specifically binds to two distinct extracellular epitopes on M(H)DM2/4 (which may, e.g., lead to the amplification of complement activation, the increased deposition of fragments (C3b, iC3b, C3d, C3g, C4b) on the cancer cell surface membrane, and/or the increased cancer cell killing by the MAC). In another embodiment, the antibody or fragment is a bispecific antibody or fragment that specifically binds to an extracellular epitope of M(H)DM2/4 and specifically binds to an extracellular epitope of a complement regulatory protein (CRP) (which may, e.g., prevent the degradation of the freshly deposited immunologically active fragments (C3b, iC3b, C3d, C3g, C4b) by CRPs, amplify the activation of the complement cascade, and/or amplify MAC induced cancer cell lysis). Such bispecific anti-M(H)DM2/4 antibodies or fragments may increase CDC activity, increase ADCC activity, increase antibody-dependent cellular phagocytosis (ADCP) by neutrophils and macrophages, or increase CDC, ADCC and ADCP.

In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate antibody-dependent cell-mediated cytoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP).

Methods of making an antibody that has ADCC function are known in the art.

Methods of making an antibody that has ADCP function are known in the art. Generally, the Fc region of the antibody mediates its binding to an Fc receptor, FcR, on neutrophils, macrophages, natural killer cells, eosinophils and mast cells, which leads to ADCC and on neutrophils, macrophages and dendritic cells resulting in ADCP. In some embodiments, in which the ADCC and/or ADCP activity is desired, the Fc region of the antibody described herein is of a human IgG (e.g., IgG1, IgG2, IgG3) type or a human IgE type. In one embodiment, the Fc region of the antibody described herein is of a human IgG1 isotype.

In one embodiment, the Fc region of the antibody described herein is bioengineered (e.g., via cross-linking, via di-sulfide bond formation, via oligosaccharide addition, or via mutation) to increase its ADCC and/or ADCP activity. In one embodiment, the Fc region of the antibody described herein is mutated to increase the lifespan of the intact antibody (e.g., in accordance with the methods described in Vaccaro et al., 2005, Nat Biotechnol. 23:1283-8128, the disclosure of which is incorporated by reference herein). In another embodiment, amino acid substitutions in the Fc CH2 and CH3 domains can be employed to direct the efficacy towards ADCC/ADCP and away from CDC or to increase the efficiency of all three cytotoxic activities (i.e., ADCC, ADCP and CDC). In another embodiment, CH2 and/or CH3 domains of the Fc region of the antibody described herein are modified at their glycosylation sites to remove/reduce fucose residues in order to improve ADCC and/or ADCP function, e.g., in accordance with the methods described in, e.g., Satoh et al., 2006, Expert Opin Biol Ther. 6:1161-1173 and/or Liu et al., 2015, Ca Immunol Res. 3:173-183, the disclosures of which are incorporated by reference herein).

In a specific embodiment, the anti-M(H)DM2/4 antibodies described herein having an IgG Fc region are bioengineered at their Fc region to change the N-glycan structure at their glycosylation site to the GO glycan type terminating in GlcNAc (N-acetylglucosamine), and without fucose and sialic acid residues (which may result in, e.g., the activation of both the classic and alternate pathway of complement pathways and in increased binding to lectins including the mannose-binding lectins secreted during inflammatory responses).

In one embodiment, the Fc region of the antibody described herein is of a human IgG1 isotype and has alanine substitution at position 333 of its CH2 domain. In one embodiment, the Fc region of the antibody described herein is of a human IgG1 isotype and has a triple mutation S239D/I332E/A330L (which leads to a higher affinity for FcγRIIIa and a lower affinity for FcγRIIb resulting in enhanced ADCC) (such Fc modification can be made, e.g., in accordance with the methods described in Lazar et al., 2006, PNAS 103:4005-4010). In one embodiment, the Fc region of the antibody described herein is of a human IgG1 isotype and has a triple mutation S239D/I332E/G236A (which leads to improved FcγRIIIa affinity and FcγRIIa/FcγRIIb ratio that mediates enhanced phagocytosis of target cells by macrophages) (such Fc modification can be made, e.g., in accordance with the methods described in Richards et al., 2008, Mol. Cancer Ther. 7:2517-27).

In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate both complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytoxicity (ADCC). In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments described herein are antibodies or fragments that mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In other embodiments, contemplated herein is an antibody or fragment that mediates only CDC or only ADCC activity. The CDC and ADCC function of the antibodies described herein can be tested by any in vitro and/or in vivo cytotoxicity assays known in the art. In certain embodiments, an antibody or fragment used herein comprises one or more amino acid mutations or substitutions in the Fc region that improve its CDC or ADCC activity (e.g., any mutations or substitutions described herein or known in the art (see, e.g., Idusogie et al., 2001. J Immunol. 166(4):2571-5; Strohl, 2009, Curr Opin Biotechnol. 20(6):685-91; Lazar et al., 2006, PNAS 103(11): 4005-4010, the disclosures of which are incorporated by reference herein)).

In a specific embodiment, the anti-M(H)DM2/4 antibodies or fragments are unconjugated, for example, are not conjugated to a cytotoxic drug).

In certain embodiments, the anti-M(H)DM2/4 antibodies or fragments described herein are not bound (e.g., not conjugated) to a drug (e.g., to a cytotoxic drug). In some of these embodiments, the anti-M(H)DM2/4 antibodies or fragments mediate CDC and/or ADCC.

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof specifically binds to an extracellularly accessible segment (i.e. epitope) within amino acids 1 to 15, 15 to 25 or 475 to 491 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of HDM2 (SEQ ID NO:4)). Amino acids of SEQ ID NO:1 (which are amino acids 1 to 15 of SEQ ID NO:4) are in an extracellularly accessible epitope of HDM2.

In one embodiment, an anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of HDM2 (SEQ ID NO:4)). Amino acids of SEQ ID NO:2 (which are amino acids 15 to 25 of SEQ ID NO:4) are in an extracellularly accessible epitope of HDM2.

In one embodiment, an anti-HDM2 antibody or fragment thereof specifically binds to HDM2 within amino acids of SEQ ID NO:3 (which are amino acids 475 to 491 of HDM2 (SEQ ID NO:4)). Amino acids of SEQ ID NO:3 (which are amino acids 475 to 491 of SEQ ID NO:4) are in an extracellularly accessible epitope of HDM2.

In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of HDM2 within amino acids 50 to 60 of M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).

In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of M(H)DM2/4 within amino acids 100 to 110 of M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).

In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellular epitope of M(H)DM2/4 within amino acids 1 to 126 of M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).

In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of M(H)DM2/4 within amino acids 436 to 482 of M(H)DM2/4 (SEQ ID NO:4 or SEQ ID NO:6).

In one embodiment, anti-M(H)DM2/4 antibodies or fragments thereof specifically bind to an extracellularly accessible epitope of M(H)DM2/4 within the terminal 100 amino acids at the C-terminus of the M(H)DM2/4 (e.g., M(H)DM2/4 protein variant (splice variant) known or expected to be expressed on the plasma membrane of cells of the cancer type being treated, or M(H)DM2/4 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

The invention also provides anti-M(H)DM2/4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:1 (e.g., NMC-103 antibody described herein, or any antibody or fragment having the VH of NMC-103 (i.e., the VH of SEQ ID NO:36) and the VL of NMC-103 (i.e., the VL of SEQ ID NO:37), or any antibody or fragment having the VH and VL CDRs of NMC-103). The invention also provides anti-M(H)DM2/4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:2 (e.g., NMC-204 antibody described herein, or any antibody or fragment having the VH of NMC-204 (i.e., the VH of SEQ ID NO:38) and the VL of NMC-204 (i.e., the VL of SEQ ID NO:39), or any antibody or fragment having the VH and VL CDRs of NMC204). The invention also provides anti-M(H)DM2/4 antibodies or fragments thereof that compete for binding to HDM2 with an antibody that specifically binds to HDM2 within the amino acid sequence of SEQ ID NO:3 (e.g., NMC-303 antibody described herein, or any antibody or fragment having the VH of NMC-303 (i.e., the VH of SEQ ID NO:40) and the VL of NMC-303 (i.e., the VL of SEQ ID NO:41), or any antibody or fragment having the VH and VL CDRs of NMC-303).

Any competition assay known in the art can be used to identify an antibody that competes with an antibody described herein for binding to M(H)DM2/4 (see Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)). In an exemplary competition assay, immobilized M(H)DM2/4 (e.g., immobilized on a microtiter plate or well) is incubated in a solution comprising a first labeled antibody that binds to M(H)DM2/4 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to M(H)DM2/4. As a control, immobilized M(H)DM2/4 can be incubated in a solution comprising the first labeled antibody but without the second unlabeled antibody. After incubation, excess unbound antibody is removed, and the amount of label associated with immobilized M(H)DM2/4 is measured. The substantial reduction of the amount of label in the test sample relative to the control sample indicates that the second antibody is competing with the first antibody for binding to M(H)DM2/4.

In specific embodiments, an antibody that competes with an antibody described herein (e.g., antibodies having the VH and VL of NMC-103, NMC-204 or NMC-303) for binding to M(H)DM2/4 also binds to the same peptide derived from M(H)DM2/4 that is bound by such antibody (e.g., the peptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3). In specific embodiments, an antibody that competes with an antibody described herein (e.g., antibodies having the VH and VL of NMC-103, NMC-204 or NMC-303) for binding to M(H)DM2/4 also binds to the same epitope in M(H)DM2/4 that is bound by such antibody.

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof binds to the same epitope of M(H)DM2/4 as an antibody or fragment having a VH of SEQ ID NO:36 and a VL of SEQ ID NO:37. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein binds to the same epitope of M(H)DM2/4 as an antibody or fragment having a VH of SEQ ID NO:38 and a VL of SEQ ID NO:39. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein binds to the same epitope of M(H)DM2/4 as an antibody or fragment having a VH of SEQ ID NO:40 and a VL of SEQ ID NO:41.

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 19 to 50 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 19 to 108 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 154 to 167 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1 to 60 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1 to 100 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 1 to 108 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 26 to 60 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within the terminal 60 amino acids at the C-terminus of the HDM2 (e.g., HDM2 protein variant (splice variant) known or expected to be expressed on the plasma membrane of cells of the cancer type being treated, or HDM2 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within the terminal 100 amino acids at the C-terminus of the HDM2 (e.g., HDM2 protein variant (splice variant) known or expected to be expressed on the plasma membrane of cells of the cancer type being treated, or HDM2 protein variant (splice variant) determined to be expressed on the plasma membrane of cancer cells of the subject being treated).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein specifically binds to an extracellularly accessible epitope within amino acids 101 to 200 of HDM2 (SEQ ID NO:4).

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody OP145 (monoclonal antibody commercially available from Calbiochem, Catalogue No. OP145-100UG; see Table 10, below, for further details regarding OP145). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody 965 (SMP14) (monoclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-965; see Tables 3 and 10, below, for further details regarding 965 (SMP14)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody sc-813 (N-20) (polyclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-813; see Table 10, below, for further details regarding sc-813 (N-20)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody sc-812 (C-18) (polyclonal antibody commercially available from Santa Cruz, Catalogue No. Sc-812; see Table 10, below, for further details regarding sc-812 (C-18)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein competes for binding to HDM2 with antibody M01, clone 1A7 (monoclonal antibody commercially available from Abnova, Catalogue No. H00004193-M01; see Table 3, below, for further details regarding M01, clone 1A7).

Any competition assay known in the art can be used to identify an antibody that competes with antibody OP145, SMP14, N-20, C-18, or M01, clone 1A7 for binding to M(H)DM2/4 (e.g., HDM2) (see Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)).

In specific embodiments, an antibody that competes with antibody OP145, SMP14, N-20, C-18, or M01, clone 1A7 for binding to M(H)DM2/4 also binds to the same epitope that is bound by such antibodies.

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody OP145 (see Table 10, below, for further details regarding OP145). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody 965 (SMP14) (see Tables 3 and 10, below, for further details regarding 965 (SMP14)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein binds to one of the same epitope(s) of HDM2 as polyclonal antibody sc-813 (N-20) (see Table 10, below, for further details regarding 813 (N-20)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein binds to one of the same epitope(s) of HDM2 as polyclonal antibody sc-812 (C-18) (see Table 10, below, for further details regarding sc-812 (C-18)). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof for use in the methods described herein binds to the same epitope of HDM2 as antibody M01, clone 1A7 (i.e., monoclonal antibody commercially available from Abnova, Catalogue No. H00004193-M01; see Table 3, below, for further details regarding M01, clone 1A7).

In a specific embodiment, the anti-M(H)DM2/4 antibody or fragment thereof described herein is purified. In certain embodiments, an antibody or fragment is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods (see Flatman et al., J. Chromatogr. B 848:79-87 (2007) for review of methods for assessment of antibody purity).

The anti-M(H)DM2/4 antibody or fragment described herein can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Such labeled antibodies or fragments can be used to detect M(H)DM2/4 on the plasma membrane surface of cells.

Examples of detectable labels or substances include enzyme labels, radioisotopes (e.g., iodine, carbon, sulfur, tritium, indium, and technetium), luminescent labels, fluorescent labels, and biotin. This methodology can be used to determine whether cells of a certain cancer (e.g., cells of cancer in a patient) express M(H)DM2/4, or a certain splice variant of M(H)DM2/4, on the plasma membrane, where the detection of M(H)DM2/4 using the antibody (or fragment) may indicate that the antibody (or fragment) (with or without the detectable label or substance) can be used in the diagnosis and treatment of the cancer or preventing metastases of the cancer.

7.2 Antibody-Drug Conjugates

In a specific embodiment, the invention provides antibody-drug conjugates comprising an anti-M(H)DM2/4 antibody or fragment described herein bound (e.g., covalently bound) to a cytotoxic drug. In such embodiments, the antibody-drug conjugates are intended to mediate cytotoxicity by delivery of a cytotoxic drug to the cells of the cancer.

Accordingly, an anti-M(H)DM2/4 antibody or an antigen-binding fragment described herein can be bound or conjugated to one or more cytotoxic agents. The cytotoxic agent can be any agent that inhibits or prevents a vital cellular function (e.g., cell division) and/or causes cell death or destruction. The cytotoxic agents that can be bound or conjugated to an anti-M(H)DM2/4 antibody or fragment include, without limitation, chemotherapeutic agents (e.g., any chemotherapeutic agent known in the art or described herein), toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes, growth inhibitory agents, and nucleolytic enzymes. The antibody-drug conjugates and their methods of making (including the types of antibodies that can be used in such conjugates, drugs that can be used in such conjugates, and linkers that can be used to link the antibody to the drug) are known in the art (see, e.g., Peters & Brown, 2015, Biosci. Rep. 35, e00225, doi:10.1042/BSR20150089).

Examples of the cytotoxic agents that can be conjugated to an anti-M(H)DM2/4 antibody or fragment described herein include, without limitation, anthracyclin, doxorubicin, methotreaxate, an anti-metabolite agent, an anti-folate agent, an auristatin (e.g., MMAE or MMAF), a maytansine, a calicheamicin, a duocarymucin, and a pyrrolobenzodiazepine (PBD) dimer.

In specific embodiments, an M(H)DM2/4 antibody or fragment described herein is conjugated to one or more of the following drugs: a maytansinoid, an auristatin (such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)), a dolastatin, a calicheamicin or derivative thereof, an anthracycline (such as daunomycin or doxorubicin), methotrexate, vindesine, a taxane (such as docetaxel), paclitaxel, larotaxel, tesetaxel, ortataxel, and a trichothecene.

In another embodiment, an M(H)DM2/4 antibody or fragment described herein is conjugated to a toxin or a fragment thereof (e.g., diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or a tricothecene).

In another embodiment, an M(H)DM2/4 antibody or fragment described herein is conjugated to a radioactive isotope (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹², or a radioactive isotope of Lu).

In another embodiment, an M(H)DM2/4 antibody or fragment thereof described herein is conjugated to nanoparticles or other targeting tools to promote concentrated delivery to and retention of the antibodies at the tumor site.

7.3 Making of Antibodies

The anti-M(H)DM2/4 antibodies or fragments described herein can be produced by any method known in the art.

Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

Methods for producing monoclonal antibodies are also known in the art, and include the use of hybridoma, recombinant and phage display technologies, and the use of humanized mice. For example, monoclonal antibodies can be produced using hybridoma techniques as taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), or in Kohler G & Milstein C (1975) Nature 256: 495. In another example, human monoclonal antibodies can be produced using humanized mice as taught, for example, in Laffleur et al., 2012, Methods Mol. Biol. 901:149-59.

Methods for producing specific antibodies using hybridoma technology are routine and well known in the art. In particular, a mouse or another appropriate host animal can be immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the target protein (i.e., extracellular region of M(H)DM2/4) used for immunization. Lymphocytes then are fused with myeloma cells to form a hybridoma cell (see Goding J W (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986); see also Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The hybridoma cells are then grown in a suitable culture medium, which can be assayed for production of monoclonal antibodies directed against M(H)DM2/4. The binding specificity of monoclonal anti-M(H)DM2/4 antibodies produced by this method can be determined by methods known in the art, e.g., immunoprecipitation or an in vitro assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The hybridoma clones thus selected are then grown by standard methods (see Goding J W (Ed), Monoclonal Antibodies: Principles and Practice, supra). The monoclonal antibodies can then be separated from the culture medium and purified.

Further, the antibodies or fragments described herein can also be made using various phage display technologies known in the art (see Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; and Burton D R & Barbas C F (1994) Advan Immunol 57: 191-280).

Methods for producing chimeric antibodies (i.e., antibody with a variable region of one species (e.g., murine) and a constant region of another species (e.g., human)) are known in the art (see Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L (1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).

Methods of making antibody fragments are known in the art. For example, Fab and F(ab′)₂ fragments can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

Methods of making humanized antibodies are also known in the art (see International Publication No. WO 91/09967; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G M et al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al., (1994) PNAS 91: 969-973; International Publication No. WO 93/17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000), Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) Protein Eng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; Sandhu J S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J Mol Biol 235(3): 959-73). In a specific embodiment, a humanized antibody is made by CDR grafting.

Methods of making human antibodies are known in the art and include phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741). In some embodiments, human antibodies can be produced using mouse-human hybridomas (see Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31).

Methods of making single domain antibodies, for example, antibodies lacking the light chains, are also known in the art (see Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302).

Methods of making single chain Fv (scFv) antibodies are also known in the art (see Ahmad et al., 2012, Clinical and Developmental Immunology, doi:10.1155/2012/980250; Wang et al., 2006, Anal. Chem. 78, 997-1004; Pansri et al., 2009, BMC Biotechnology 9:6). For example, scFv antibodies can be constructed by fusing variable domains of heavy and light chains of immunoglobulins via short polypeptide linkers (using recombinant expression techniques), and scFv antibodies having desired antigen-binding properties can be selected by phage display technology.

Methods of producing bispecific antibodies are well known in the art (Konterman, 2012, MAbs 4:182-197; Gramer et al., 2013, MAbs 5:962-973).

Methods of conferring CDC or ADCC activity on an antibody (such as an antibody that does not have CDC or ADCC activity to begin with) are known in the art (see Kellner et al., 2014, Methods 65:105-113; International Publication No. WO 2012010562; Natsume et al., 2009, Drug Design, Development and Therapy 3(3):7-16). Such methods include, without limitation, Fc region isotype shuffling, amino acid mutations in the Fc region conferring enhanced or optimized CDC and/or ADCC activity, and changes in the Fc region glycosylation profile conferring enhanced or optimized CDC and/or ADCC activity).

7.4 Antibody Selection

If a candidate antibody or fragment for use in the therapeutic and diagnostic methods provided herein is not yet known or has not yet been demonstrated to bind to a region of M(H)DM2/4 exposed on the surface of cancer cells, the antibody or fragment may optionally be tested by any of the following methods:

In certain aspects, provided herein is a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein (e.g., for diagnosis of cancer, treating cancer or for preventing metastases) comprising: (a) contacting intact cancer cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells, in particular on the extracellular surface of the cancer cells (e.g., relative to intact cancer cells not contacted by said antibody or fragment), wherein the binding of the anti-M(H)DM2/4 antibody or fragment to the intact cancer cells contacted with such antibody or fragment indicates that said antibody or fragment is suitable for use in the methods described herein. In a specific embodiment, the cancer cells are from the patient proposed to be treated with the antibody or fragment thereof.

In certain aspects, provided herein is a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein (e.g., for diagnosis of cancer, treating cancer or for preventing metastases) comprising: (a) contacting intact cancer cells (e.g., cancer cells expected, known, or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the antibody or fragment binds to the intact cancer cells (in particular on the extracellular surface of the cancer cells) at an increased level relative to binding to intact normal cells (e.g., non-cancerous cells of the same tissue or organ type as the cancer cells), wherein increased binding of the anti-M(H)DM2/4 antibody or fragment to the intact cancer cells relative to normal cells indicates that said antibody or fragment is suitable for use in the methods described herein.

In certain aspects, provided herein is a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein (e.g., for treating cancer or for preventing metastases) comprising: (a) contacting intact cancer cells (e.g., cancer cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the contacting step results in an increased amount of cell death or destruction of the intact cancer cells (e.g., as determined by cell-death markers such as Propidium Iodide staining) relative to the amount of death or destruction of intact cancer cells not contacted by said antibody or fragment and/or relative to the amount of death or destruction of intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted by said antibody or fragment, wherein increased amount of cell death or destruction of the intact cancer cells contacted with the antibody or fragment indicates that said antibody or fragment is suitable for use in the methods described herein.

In certain aspects, provided herein is a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein (e.g., for treating cancer or for preventing metastases) comprising: (a) contacting intact cancer cells (e.g., cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or fragment; and (b) determining whether the contacting step results in an increased level of complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (as determined by one or more cytotoxicity assays) towards the intact cancer cells relative to the level of CDC or ADCC towards intact cancer cells not contacted by said antibody or fragment and/or relative to the level of CDC or ADCC towards intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted by said antibody or fragment, wherein increased level of CDC or ADCC towards the intact cancer cells indicates that said antibody or fragment is suitable for use in the methods described herein.

In certain aspects, provided herein is a method for identifying an anti-M(H)DM2/4 antibody or a fragment thereof for use in the methods described herein (e.g., for treating cancer or for preventing metastases) comprising: (a) contacting intact cancer cells (e.g., cells known, expected or determined to express M(H)DM2/4) with an anti-M(H)DM2/4 antibody or a fragment thereof; and (b) determining whether the contacting step results in increased inhibition of growth and proliferation of the intact cancer cells relative to the inhibition of growth and proliferation of intact cancer cells not contacted by said antibody or fragment and/or relative to the inhibition of growth and proliferation of intact normal cells (e.g., non-cancerous cells of the same tissue or organ as the cancer cells) contacted by said antibody or fragment, wherein increased inhibition of growth and proliferation of the intact cancer cells contacted with the antibody or fragment indicates that said antibody or fragment is suitable for use in the methods described herein.

7.5 Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising an anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. Also provided herein are pharmaceutical compositions comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier. Appropriate pharmaceutically acceptable carriers including, but not limited to, excipients and stabilizers) are known in the art (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). The anti-M(H)DM2/4 antibody or fragment, or antibody-drug conjugate, in the pharmaceutical compositions described herein can be purified.

Pharmaceutically acceptable carriers may include an isotonic agent, a buffer, a suspending agent, a dispersing agent, an emulsifying agent, a wetting agent, a sequestering or chelating agent, a pH buffering agent, a solubility enhancer, an antioxidant, an anesthetic, and/or an antimicrobial agent. Suitable excipients include, without limitation, water, saline, glycerol, ethanol, starch, lactose, sucrose, gelatin, malt, propylene, silica gel, sodium stearate, base cream and dextrose. If administered parenterally, suitable pharmaceutically acceptable carriers may include physiological saline or phosphate buffered saline (PBS), and solutions containing such agents as glucose, polyethylene glycol, polypropylene glycol or other agents.

In specific embodiments, pharmaceutical compositions provided herein comprise an anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof, or antibody-drug conjugate, described herein, and optionally one or more other therapeutic (e.g., anti-cancer) agents, in a pharmaceutically acceptable carrier.

A pharmaceutical composition may be formulated for any route of administration to a subject. Formulations for injections can be prepared as liquid solutions, suspensions, emulsions, or solid forms suitable for making into a solution or suspension prior to injection.

The anti-M(H)DM2/4 antibody or fragment thereof, or antibody-drug conjugate, can be used or present in the pharmaceutical composition in a therapeutically effective amount. The therapeutically effective amount of the antibody or conjugate can be determined by standard clinical techniques.

In a specific embodiment, a pharmaceutical composition contemplated for use in the therapeutic methods described herein comprises an anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof, or antibody-drug conjugate described herein, and does not comprise any additional anti-cancer agent or therapy. In another specific embodiment, a pharmaceutical composition contemplated for use in the therapeutic methods described herein comprises an anti-M(H)DM2/4 antibody or an antigen-binding fragment thereof, or antibody-drug conjugate described herein, and further comprises an additional anti-cancer agent or therapy (e.g., any one, two or more additional anti-cancer agents or therapies described herein).

In one aspect, provided herein is a vaccine composition comprising: (i) an immunogenic amount of a peptide, wherein the amino acid sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3), or a polynucleotide encoding the peptide; and (ii) a pharmaceutically acceptable carrier. In a certain embodiment, provided herein is a vaccine composition comprising: (i) an immunogenic amount of a peptide, wherein the amino acid sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3); and (ii) a pharmaceutically acceptable carrier. In one embodiment of the vaccine compositions described herein, the peptide is purified. In one embodiment of the vaccine compositions described herein, the composition further comprises an adjuvant (such as an immune-stimulating adjuvant). In one embodiment of the vaccine compositions described herein, the pharmaceutically acceptable carrier is a liposome. In one aspect, provided herein is a method of vaccinating a subject at risk for developing cancer or a subject who has been diagnosed with cancer by administering to the subject the vaccine composition described herein. A vaccine composition can be produced using any method known in the art (see, e.g., Li et al., 2014, Vaccines 2:515-536).

7.6 Therapeutic Methods

In one aspect, the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression, and/or increasing survival) in a subject in need thereof comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In a specific embodiment, the invention provides a method of treating cancer in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4. In another specific embodiment, the invention provides a method of preventing metastasis in a subject in need thereof, said method comprising administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4. In a specific embodiment of the methods described herein, the antibody or fragment is not bound to a cell-penetrating peptide.

In one aspect, the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression, and/or increasing survival) in a subject in need thereof comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellular region of HDM2 exposed on their plasma membrane surface), preferably wherein the antibody or fragment is not bound to a cell-penetrating peptide. In one embodiment, the method of treating cancer encompasses preventing metastasis of the cancer. In one embodiment, the method of treating cancer is a method for reducing tumor size (as measured, e.g., by tumor volume or diameter), inhibiting the growth of the tumor, reducing the growth of the tumor, or eradicating the tumor.

In another aspect, the invention provides for preventing metastases in a subject that has cancer comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In another aspect, the invention provides for preventing metastases in a subject that has cancer comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellular region of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellular region of HDM2 exposed on their plasma membrane surface).

In another aspect, the invention provides for reducing the number, size or invasiveness of metastases, or eradicating metastases, in a subject that has a metastatic cancer comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In another aspect, the invention provides for reducing the number, size or invasiveness of metastases, or eradicating metastases, in a subject that has a metastatic cancer comprising administering to the subject an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of HDM2 exposed on the surface of cancer cells (e.g., where the cells of the type of cancer being treated are known or expected to have such extracellularly accessible epitope of HDM2 exposed on their plasma membrane surface).

In one aspect, the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression, and/or increasing survival) in a subject who has experienced an accelerated rate of cancer growth in response to administration of an inhibitor of one or more inhibitory checkpoint molecules, comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In one aspect, the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression, increasing survival) in a subject who has experienced hyper-progression in response to administration of an inhibitor of one or more inhibitory checkpoint molecules, comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In one aspect, the invention provides for treating cancer (e.g., inhibiting cancer proliferation, inhibiting cancer progression, increasing survival) in a subject who is at risk for hyper-progression in response to administration of an inhibitor of one or more inhibitory checkpoint molecules, comprising administering to the subject any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein. In a specific embodiment, the methods described herein comprise administering to the subject: an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4. In a specific embodiment of the methods described herein, the antibody or fragment is not bound to a cell-penetrating peptide. In certain embodiments of the methods described herein, the inhibitor of one or more inhibitory checkpoint molecules is an inhibitor, such as an inhibitory antibody to, of one or more of: CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, OX40, and LAG-3. In a specific embodiment of the methods described herein, the inhibitor of one or more inhibitory checkpoint molecules is an antibody that is nivolumab (Opdivo, Bristol-Myers Squibb), pembrolizumab (Keytruda, Merck), avelumab (Bavencio; EMD Serono, Pfizer), atezolizumab (Tecentriq, Genentech), durvalumab (Imfinzi, AstraZeneca), cemiplimab (Libtayo, Regeneron) or ipilimumab (Yervoy, Bristol-Myers Squibb). In one embodiment, the inhibitor of one or more inhibitory checkpoint molecules is an inhibitor of, such as an inhibitory antibody to, PD-1. In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, identifying the subject as a hyper-progressor in response to administration of an inhibitor of one or more inhibitory checkpoint molecules (and, e.g., administering any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the subject is a hyper-progressor). In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining whether any anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein binds to the surface of intact cells of the cancer (e.g., intact cancer cells isolated from the subjects) (and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein binds to the surface of intact cells of the cancer). In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining whether the subject has a gene amplification of M(H)DM2/4 (and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the subject has a gene amplification of M(H)DM2/4). In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining how many copies of the M(H)DM2/4 gene are present in the cancer cells of the subject, and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the cancer cells of the subject have 8 or more than 8 copies of M(H)DM2/4 gene. In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining the degree of M(H)DM2/4 gene amplification in the cancer cells of the subject, and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the cancer cells of the subject have 50% or more than 50% amplification of the M(H)DM2/4 gene relative to the normal cells of the subject (or relative to the cancer cells of the subject prior to administration of an inhibitor of an immune checkpoint molecule). In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2. In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM4. In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2 and HDM4. In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining whether the subject has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells (and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the subject has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells). In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining whether the subject has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in cancer cells prior to administration of the inhibitor of a checkpoint molecule (and, e.g., administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the subject has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in cancer cells prior to administration of the inhibitor of a checkpoint molecule). In certain embodiments, the methods described herein further comprise, before administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein, determining whether the subject has an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to its binding to the surface of intact normal cells (or relative to its binding to the surface of intact cells of the cancer prior to administration of an inhibitor of an immune checkpoint molecule). In certain embodiments, the methods described herein comprise administering an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein if the subject has an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to its binding to the surface of intact normal cells (or relative to its binding to the surface of intact cells of the cancer prior to administration of an inhibitor of an immune checkpoint molecule). In certain embodiments of the methods described herein, the cancer is a bladder cancer, a breast cancer (e.g., a triple negative breast cancer), a lung cancer, a colon cancer, a melanoma, a sarcoma (e.g., an endometrial stromal sarcoma), an ovarian cancer, a glioma, a head and neck cancer, an urothelial cancer, a pancreatic cancer, or a squamous cell carcinoma of the hypopharynx. In certain embodiments of the methods described herein, an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein is administered in combination with another therapy, such as immunotherapy (e.g., an inhibitor of an inhibitory checkpoint molecule), a chemotherapy, a small molecule inhibitor (such as an inhibitor of one or more of: EGFR, KRAS, STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK, PI3K/AKT, TGF-β, DNMT3A, IFNγ, JAK1/JAK2/JAK3, CD274, PTEN, ART, CDK), or an inhibitor of p53-H(M)DM2 interaction (e.g., a small molecule inhibitor or a peptide inhibitor). In certain embodiments of the methods described herein, an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein is administered as a monotherapy.

In one aspect, the invention provides for treatment of a cancer that is resistant to another cancer therapy or therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody). In one embodiment, the invention provides for treating a cancer resistant to chemotherapy (i.e., resistant to one or more chemotherapeutic drugs). In one embodiment, the invention provides for treating a cancer resistant to treatment with another monoclonal antibody or antibodies. In one embodiment, the invention provides for treating a cancer resistant to radiotherapy. In one embodiment, the invention provides for treating a cancer resistant to small molecule targeted therapy or therapies.

Disclosed herein is therapeutic use of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or antigen-binding fragment thereof in a patient who has cancer (e.g., has been diagnosed with cancer). In preferred embodiments, disclosed herein is therapeutic use of an anti-HDM2 antibody (or a fragment thereof) in a patient who has cancer that is known to metastasize (i.e., is a type of cancer that is commonly known to become metastatic cancer). In some embodiments, the patient being treated has metastatic cancer. In other embodiments, a patient with a cancer that has not metastasized is treated in accordance with a method described herein in order to prevent metastasis of the cancer. In some embodiments, the patient being treated has been previously treated with other cancer therapies (e.g. vaccine, targeted therapy, chemotherapy, immunotherapy). In a specific embodiment, the patient with a cancer that has metastasized is treated in accordance with a method described herein in order to reduce, slow down or stop metastases, or decrease the number or size of metastases of the cancer.

The methods described herein are suitable for treating cancers that are expected, known or determined to express anti-M(H)DM2/4 (e.g., HDM2) on the surface of their cells. The HDM2 on the surface of cancer cells targeted by the methods described herein can be one of the splice variants of HDM2 protein known in the art or described herein. Without intending to be bound by a mechanism of action, it is believed that HDM2 on the surface of cancer cells is usually a splice variant of the HDM2 protein that lacks at least one or all nuclear localization signals (e.g., the nuclear localization sequence at the N-terminal portion of HDM2, the nuclear localization signal at the C-terminal portion of HDM2, or both nuclear localization signals). In a specific embodiment, HDM2 on the surface of cancer cells is a splice variant of the HDM2 protein that lacks at least one or all nuclear localization signals, and further lacks a nuclear export signal. In one embodiment, the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signal at amino acids 179-185 of SEQ ID NO: 4 (i.e., lacks amino acids 179 to 185 of SEQ ID NO: 4). In one embodiment, the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signal at amino acids 466-473 of SEQ ID NO: 4 (i.e., lacks amino acids 466 to 473 of SEQ ID NO: 4). In one embodiment, the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear localization signals at amino acids 179-185 and amino acids 466-473 of SEQ ID NO: 4 (i.e., lacks amino acids 179 to 185 and 466 to 473 of SEQ ID NO: 4). In some embodiments, the HDM2 on the surface of cancer cells is HDM2 that lacks the nuclear export signal, such as the nuclear export signal at amino acids 190-202 of SEQ ID NO: 4 (i.e., lacks amino acids 190 to 202 of SEQ ID NO: 4).

In certain embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein, such as one of the splice variants known in the art or described herein (see Table 1 and Table 2, and Section 8, for the list of HDM2 variants). In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-A (SEQ ID NO: 8), which lacks amino acids 28-222 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-A1 (SEQ ID NO: 9), which lacks amino acids 28-222 and 275-300 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-B (SEQ ID NO: 10), which lacks amino acids 28-300 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-C(SEQ ID NO: 11), which lacks amino acids 53-222 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-D (SEQ ID NO:12), which lacks amino acids 30-388 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-E (SEQ ID NO: 13), which lacks amino acids 76-102 and 103-491 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-F (SEQ ID NO: 14), which lacks amino acids 53-97 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-G (SEQ ID NO: 15), which lacks amino acids 115-169 of SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-11 (SEQ ID NO: 16), in which amino acid M has been substituted with M→MVRSRQM in SEQ ID NO: 4. In some embodiments, the HDM2 on the surface of cancer cells targeted by the methods described herein is a splice variant of the HDM2 protein known as MDM2-KB2 (SEQ ID NO: 17), which lacks amino acids 157-248 of SEQ ID NO: 4. In some embodiments, the methods described herein target a splice variant of M(H)DM4 on the surface of cancer cells, for example, target one or more of the following splice variants: MDMX-S, MDM4-S, MDM4-A, MDM4-G, MDM4-XALT1/XALT2 and MDM4-211 (or a human equivalent of the listed splice variants).

In a specific embodiment, the anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or a fragment thereof for use in the methods described herein has been tested and determined to be expected to bind to HDM2 exposed on the surface of the cells of the cancer to be treated. This binding to HDM2 can be shown by, for example, the ability of the anti-HDM2 antibody or fragment to bind to an intact cancer cell of the tissue type of the tissue of origin of the cancer being treated (which can be but does not need to be from the subject being treated). In one embodiment, an anti-HDM2 antibody or a fragment thereof is tested and determined to be expected to bind to the intact cells of the cancer of a subject, before administering the antibody or fragment to the subject. This testing can be done, e.g., by showing binding of the anti-HDM2 antibody or fragment to the surface of intact cancer cells obtained by biopsy or to a cancer cell line of the appropriate tissue type.

Optionally, the cancer cells of the prospective patient to be treated can be tested for expression of M(H)DM2/4 (e.g., HDM2) on their surface using techniques known in the art in order to determine whether the subject is an appropriate candidate for anti-HDM2 therapy described herein; however, such ordinarily would not be deemed necessary if the patient has a cancer of a tissue type that is known or expected to have an extracellularly accessible epitope of M(H)DM2/4. In a specific embodiment, the cells of the cancer in the subject being treated have been tested and determined to have an extracellularly accessible epitope of HDM2 (targeted by the anti-HDM2 antibody or fragment thereof) exposed on their plasma membrane surface (e.g., determined to express a variant of HDM2 that is known to have this extracellular region exposed on the plasma membrane surface). In certain embodiments, the cancer being treated using the methods described herein is a cancer that is known or determined to express a splice variant of HDM2 (for example, MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C(SEQ ID NO: 11), MDM2-D (SEQ ID NO:12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16) or MDM-KB2 (SEQ ID NO: 17)), on the plasma membrane surface of its cells. Such can be detected using techniques known in the art (e.g., RT-PCR on a nucleic acid sample from cancer biopsy).

In specific embodiments, the administration of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or an antigen-binding fragment thereof in accordance with the methods described herein can be carried out to achieve, or found to result in achieving, at least one, two, three, four or more of the following effects (e.g., in a subject with a cancerous tumor): (i) a decrease in tumor size (e.g., volume or diameter), (ii) a reduction in the growth of the tumor, (iii) inhibition of the progression of tumor growth, (iv) the regression of the tumor, (v) inhibition of a recurrence of the tumor, (vi) eradication of the tumor (e.g., primary tumor or metastatic tumor), (vii) prevention of metastasis of the tumor, (vii) reduction in the number, size or invasiveness of the metastases of the tumor, (viii) reduction or amelioration of the severity or duration of one or more symptoms of the tumor, (ix) the inhibition of the development or onset of one or more symptoms associated with cancer, (x) the enhancement or improvement of the therapeutic effect of another therapy, (xi) reduction in hospitalization (e.g., length of the hospitalization) in the subject, (xii) improvement in subject's quality of life, (xiii) a reduction in mortality, (xiv) an increase in a relapse free survival or the length of remission in the subject. Any of these effects can be assessed by any method known in the art. For example, the tumor size can be assessed using magnetic resonance imaging (MM), dynamic contrast-enhanced MM (DCE-MRI), X-ray, computed tomography (CT) scan, or positron emission tomography (PET) scan.

In certain embodiments, the administration of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat cancer in a subject (e.g., reduces tumor volume or diameter, reduces tumor growth, reduce tumor proliferation, eradicates the tumor, or improves one or more symptoms of cancer), when used alone or in combination with another therapy. In certain embodiments, the administration of an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to prevent metastases in a subject that has cancer, when used alone or in combination with another therapy. In certain embodiments, the administration of an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat a metastatic cancer (e.g., reduces the number, size or invasiveness of metastases, or eradicates metastases), when used alone or in combination with another therapy.

In particular embodiments, the administration of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat cancer or prevent metastasis in a subject when used alone (i.e., without an additional therapy). In other particular embodiments, the administration of an anti-HDM2 antibody or an antigen-binding fragment thereof in accordance with the methods described herein is effective to treat cancer or prevent metastasis in a subject when used in combination with one or more of the additional therapies described herein.

The effectiveness of therapies described herein can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapies described herein to the subject being treated. Alternatively, the effectiveness of therapy can be assessed by evaluating a parameter (e.g., tumor size) before and after administration of the therapies described herein to an animal model (e.g., in an animal model, such as a mouse model, a rat model, or a hamster model, of the cancer being treated). Any assay known in the art can be used to evaluate the therapeutic effectiveness of the therapies described herein.

In the therapeutic methods described herein using an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or an antigen-binding fragment thereof, it will be understood that an antibody-drug conjugate described herein can alternatively be used.

7.6.1 Cancers to be Treated

Examples of cancers that can be treated in accordance with the methods described herein include, but are not limited to, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, pancreatic cancer, skin cancer (e.g., melanoma), prostate cancer (e.g., hormone refractory, such as castration resistant, prostate cancer), lung cancer (e.g., small-cell lung cancer, or non-small cell lung cancer), colorectal cancer (e.g., colon cancer, or rectal cancer), gastrointestinal cancer, stomach cancer, small bowel cancer, appendix cancer, esophageal cancer, gastric cancer, renal cancer, bladder cancer, gallbladder cancer, kidney cancer (e.g., renal cell carcinoma, or Wilms tumor)), liver cancer (e.g., hepatic carcinoma, or hepatoma), central nervous system cancer (e.g., brain cancer), peripheral nervous system cancer, bronchial cancer, cancer of the oral cavity or pharynx (e.g., oropharyngeal cancer, laryngeal cancer), thyroid cancer, biliary tract cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, vulvar cancer, testicular cancer, urethral cancer, vaginal cancer, penile cancer, bone cancer, eye cancer (e.g. retinoblastoma or uveal melanoma), and head and neck cancer (e.g., head and neck squamous cell carcinoma). In specific embodiments, the cancer is cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, breast cancer, or colon cancer. In one embodiment, the cancer is a pancreatic cancer. In one embodiment, the cancer is a melanoma. In one embodiment, the cancer is a breast cancer. In one embodiment, the cancer is an ovarian cancer.

In certain embodiments, the cancer that can be treated in accordance with the methods described herein is resistant to another cancer therapy or therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)). In one embodiment, the cancer that can be treated in accordance with the methods described herein is resistant to chemotherapy. In one embodiment, the cancer that can be treated in accordance with the methods described herein is resistant to treatment with another monoclonal antibody or antibodies. In one embodiment, the cancer that can be treated in accordance with the methods described herein is resistant to radiation. In one embodiment, the cancer that can be treated in accordance with the methods described herein is resistant to small molecule targeted therapy.

In a specific embodiment, the cancer treated in accordance with the methods described herein is a solid cancer. In another specific embodiment, the cancer treated in accordance with the methods described herein is a non-solid cancer (e.g., hematologic cancer).

In specific embodiments, the cancer treated in accordance with the invention is leukemia (e.g., acute leukemia (such as acute lymphocytic leukemia or acute myelocytic leukemia), chronic leukemia (such as chronic myelocytic leukemia or chronic lymphocytic leukemia), or hairy cell leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or T-cell lymphoma).

In specific embodiments, the cancer treated in accordance with the invention is carcinoma (e.g., adenocarcinoma, basal cell carcinoma, renal cell carcinoma, squamous cell carcinoma, osteocarcinoma, thyoma/thymic carcinoma, or choriocarcinoma), blastoma, sarcoma (e.g., soft tissue sarcoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, or synovia sarcoma), lymphoma, leukemia, a germ cell tumor, myeloma (e.g., multiple myeloma), squamous cell cancer, mesothelioma, glioblastoma (e.g., glioblastoma multiforme), glioma, neuroblastoma, melanoma, astrocytoma, medulloblastoma, hepatoma, seminoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, neuroma, oligodendroglioma, meningioma, or retinoblastoma.

In specific embodiments, the cancer treated in accordance with the methods described herein is a sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, endometrial stromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterine sarcoma, Kaposi sarcoma, merkel cell carcinoma, urothelial carcinoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, embryonal carcinoma, lung carcinoma (e.g., small cell lung carcinoma), bladder carcinoma, or epithelial carcinoma.

In certain embodiments, the cancer treated in accordance with the methods of the invention is metastatic. In some embodiments, the cancer treated in accordance with the methods described herein is a metastatic melanoma, a metastatic ovarian cancer, a metastatic cervical cancer, a metastatic endometrial cancer, a metastatic pancreatic cancer, a metastatic breast cancer, a metastatic colon cancer, or a metastatic brain cancer.

7.6.2 Methods of Administration

The anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments described herein (and pharmaceutical compositions comprising such antibodies) can be administered to a subject by any suitable means which include, but are not limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, intraosseous, intracerebral, intracerebroventricular, intrathecal, subcutaneous), intraperitoneal, intratumoral, intrapulmonary, intradermal, transdermal, conjunctival, intraocular, intranasal, intratracheal, oral and local intralesional routes of administration. In certain embodiments, the anti-HDM2 antibodies or fragments described herein are administered intravenously, intraarterially, intramuscularly, intraperitoneally, intratumorally, or subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intravenously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intraperitoneally. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intramuscularly. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered subcutaneously. In one embodiment, the anti-HDM2 antibodies or fragments described herein are administered intratumorally (such as by an injection into the tumor of the cancer being treated). In particular embodiments, the anti-HDM2 antibodies or fragments described herein are administered intravenously, intraperitoneally, or intratumorally.

In a specific embodiment, nano-particles coated with an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or a fragment thereof described herein are used for tumor targeting and treatment (see, for example, Cardoso et al., 2012, Curr. Med. Chem. 19(19):3103-27 and Arruebo et al., 2009, J. of Nanomater. 2009:Article ID 439389, regarding nano-particle coating with antibodies). In one embodiment, provided herein are methods for treating cancer or preventing metastasis in a subject having a cancer comprising administering to the subject nano-particles coated with an an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or a fragment thereof.

Various dosing schedules of the anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments described herein (and pharmaceutical compositions comprising such antibodies) are contemplated including single administration or multiple administrations over a period of time. The methods of administration include, without limitation, bolus administration, pulse infusions, and continuous infusions.

The therapeutic regimen for use in the methods described herein may include administration of anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments thereof (and compositions comprising such antibodies) once every week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks or once every twelve weeks (e.g., such that the subject receives from at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten doses of the antibody, or from two to twenty doses of the antibody). In certain embodiments, anti-HDM2 antibodies or fragments thereof (and compositions comprising such antibodies) are administered daily, every other day, or two, three, or four times a week (e.g., for a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two months or three months). The treatment regimens contemplated herein include regimens wherein the initial higher dose of the antibody may be followed by one or more lower doses, or wherein the initial lower dose of the antibody is followed by one or more higher doses. An exemplary treatment course (in which the anti-HDM2 antibody or fragment is administered) may last for one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, five months, six months, one year, or over several years.

In some embodiments, the initial treatment period (where the antibody is administered, e.g., once a month, once in two weeks, once a week, twice a week or three times a week) is followed by a withdrawal period in which the antibody is not administered (for, e.g., a week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months or one year), and then followed by a second treatment period (where the antibody is administered, e.g., once a month, once in two weeks, once a week, twice a week or three times a week). Such initial treatment and such second treatment periods can last, for example, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, or six months (where the initial treatment period can be the same or different from the second treatment period). This course of treatment (having the initial treatment period, a withdrawal period and a second treatment period) can be repeated twice, three times, four times, five times, six times, ten times or more than ten times.

In some embodiments, two or more antibodies or fragments thereof with different binding specificities for M(H)DM2/4 (e.g., HDM2) are administered simultaneously or sequentially to the subject being treated.

The appropriate dosage of anti-M(H)DM2/4 (e.g., anti-HDM2) antibodies or fragments for use in the methods described herein will depend on the type of antibody used, the type of cancer being treated, the severity of the cancer being treated, the route of administration, the target site, the condition of the patient (e.g., age, body weight, health), the responsiveness of the patient to the antibody, other medications used by the patient, and other factors to be considered at the discretion of the medical practitioner performing the treatment.

In certain embodiments, the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 μg/kg to 200 mg/kg of the patient's body weight. In certain embodiments, the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 μg/kg to 100 mg/kg of the patient's body weight (e.g., from about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg, or from about 0.5 mg/kg to about 100 mg/kg). In certain embodiments, the dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or fragment described herein which is administered to the subject can be from about 1 mg/kg to 200 mg/kg of the patient's body weight. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 0.025 mg/kg to about 5 mg/kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 0.05 mg/kg to about 2 mg/kg. In one embodiment, the dosage of an anti-HDM2 antibody or fragment described herein which is administered to the subject is from 5 mg/kg to about 30 mg/kg. In specific embodiments, doses (e.g., one or more doses) of about 0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg of an anti-HDM2 antibody or fragment described herein can be administered to the subject being treated. In one embodiment, a dose (e.g., one or more doses) of about 0.1 mg/kg of an anti-HDM2 antibody or fragment described herein can be administered to the subject being treated (e.g., when the antibody is administered intratumorally).

7.7 Diagnostic, Companion Diagnostic and Prognostic Methods

In one aspect, provided herein are methods of diagnosing cancer in a subject (e.g., a human), said method comprising: (a) detecting whether an antibody or a fragment thereof (e.g., a labeled antibody or fragment) that specifically binds to M(H)DM2/4 (e.g., HDM2) binds to the surface of an intact cell of the subject, wherein the antibody or fragment is any anti-M(H)DM2/4 antibody or fragment described herein (in particular, any antibody or fragment that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3); and (b) diagnosing the subject with cancer if the binding is detected in step (a). In one embodiment, the method of diagnosing further comprises, before the detecting in step (a), obtaining the intact cell from the subject, and then performing the detecting by, e.g., determining whether a labeled antibody or fragment binds to the intact cell from the subject using, e.g., FACS or cell-based ELISA analysis. In one embodiment, the method of diagnosing comprises administering the antibody or fragment to the subject before the detecting in step (a), and wherein the detecting is performed by in vivo imaging of the subject.

In one aspect, a patient is selected for treatment using an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or antibody fragment described herein based on the detection of binding of such antibody or fragment to the surface of intact cancer cells obtained from the patient. In certain embodiments, provided herein is a method of selecting a patient having a cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), wherein the detection of binding indicates that the patient can be treated with the antibody or fragment. In specific embodiments, provided herein is a method of selecting a patient having a cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, administering the antibody or fragment to the patient. The antibody or fragment administered to the patient can be the same or different from the antibody or fragment used for selection of the patient to be treated. In one embodiment, provided herein is a method of selecting a patient having an ovarian cancer for treatment with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: obtaining an intact ovarian cancer cell from the patient, determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient, and, if the binding is detected, administering the antibody or fragment to the patient. In one embodiment, any antibody or a fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used for such patient selection and/or treatment. In one embodiment, any antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, which is not bound to a cell-penetrating peptide is used for such patient selection and/or treatment. In one embodiment, any antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, which is not bound to a cell-penetrating peptide is used for such patient selection, and any antibody or a fragment thereof that specifically binds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is used for the treatment of the patient. In one aspect, provided herein are methods of diagnosing a hyper-progressive disease (such as diagnosing a subject as exhibiting hyper-progression, or likely to exhibit hyper-progression, of a cancer in response to administration of an inhibitor of an inhibitory checkpoint molecule) in subject (e.g., a human) who has a cancer, said method comprising: (a) optionally, determining whether a gene amplification of M(H)DM2/4 is present in the cells of the cancer of the subject; (b) determining whether an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer (e.g., determining whether there is an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to the binding of the antibody or fragment to the surface of intact normal cells; or determining whether there is an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to the binding of the antibody or fragment to the surface of intact cells of the cancer prior to administration of an inhibitor of an inhibitory checkpoint molecule); and (c) diagnosing the subject with the hyper-progressive disease if binding is detected in step (b), and, optionally if the gene amplification is determined to be present in step (a). In one embodiment, the method of diagnosing further comprises, before step (b), obtaining (e.g., isolating) the intact cell from the subject, and then performing the determining in step (b) by, e.g., determining whether a labeled antibody or fragment binds to the intact cell from the subject using, e.g., FACS or cell-based ELISA analysis. In one embodiment, the method of diagnosing comprises administering the antibody or fragment to the subject before step (b), and wherein the determining in step (b) is performed by in vivo imaging of the subject. In one embodiment, the determining in step (a) is performed using PCR, Next Generation Sequencing or RNA sequencing. In certain embodiments, the gene amplification is determined to be present in step (a) if the cancer cells of the subject have 8 or more than 8 copies of M(H)DM2/4 gene. In certain embodiments, the gene amplification is determined to be present in step (a) if the cancer cells of the subject have 50% or more than 50% amplification of the M(H)DM2/4 gene relative to the normal cells of the subject (or relative to the cells of the subject prior to administration of an inhibitor of an inhibitory checkpoint molecule). In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2. In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM4. In one embodiment, the gene amplification of M(H)DM2/4 is gene amplification of HDM2 and HDM4.

Non-limiting exemplary samples that can be used for in diagnostic or patient selection methods using an anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof described herein include: tissue biopsies, intact cells obtained from malignant tissues, and circulating cancer cells isolated from blood. For example, a tissue sample can be obtained from a patient and immunohistochemistry can be performed to detect whether a labeled anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to the tissue sample. Alternatively, intact cells (known or suspected to be malignant) can be isolated from a patient and FACS or cell-based ELISA analysis can be performed to detect whether a labeled anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to such cell. In yet another example, a blood sample with circulating cancer cells can be obtained from a patient and FACS or cell-based ELISA analysis can be performed to detect whether a labeled anti-M(H)DM2/4 antibody (e.g., anti-HDM2 antibody) or fragment thereof binds to such cells.

In one aspect, the duration of treatment and/or dosage of an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or antibody fragment described herein to be used in the treatment of a patient is determined based on the detection of binding of such antibody or fragment to the surface of intact cancer cells obtained from the patient. In certain embodiments, provided herein is a method of determining whether to continue the treatment of a patient having a cancer with an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 comprising: administering the antibody or fragment to the patient for a first period of time (e.g., where the patient had been selected for treatment as described above), obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, continuing administering the antibody or fragment to the patient for a second period of time (but, e.g., if the binding is not detected, discontinuing the treatment). In certain embodiments, provided herein is a method of determining whether to increase the dose of an antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2 for use in the treatment of a patient having a cancer comprising: administering a dose the antibody or fragment to the patient for a period of time (e.g., where the patient had been selected for treatment as described above), obtaining an intact cancer cell from the patient (e.g., by biopsy of the cancerous tumor in the patient, or by obtaining a blood sample with circulating cancer cells from the patient), and determining whether the antibody or fragment binds to the surface of the intact cancer cell of the patient (using any method known in the art or described herein, e.g., using cell-based ELISA or FACS analysis), and, if the binding is detected, administering a dose of the antibody or fragment to the patient for a second period of time, wherein the dose administered during the second period of time is higher than the dose administered during the first period of time (e.g., two times, or three times higher) (but, e.g., if the binding is not detected, discontinuing the treatment or administering a lower dose of the antibody or fragment during the second period of time).

In another specific embodiment, treatment with an anti-M(H)DM2/4 (e.g., anti-HDM2) antibody or an antibody fragment described herein is monitored in a patient by determining the amount of HDM2 expressed on the surface of cancer cells obtained from the patient, before and after treatment, wherein a decrease in the amount is a positive prognosis.

7.8 Patient Populations

The patients or subjects being treated in accordance with the methods described herein include, but are not limited to, humans and non-human vertebrates. In certain embodiments, the subject being treated is a mammal, e.g., a human, a dog, a cat, a monkey, a rabbit, a cow, a horse, a goat, a sheep, or a pig. In a preferred embodiment, the subject being treated is a human.

In certain embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a cancer (e.g., using a biopsy or any another method known in the art). In particular embodiments, the subject being treated has been diagnosed with an early stage cancer. In other embodiments, the subject being treated has been diagnosed with an advanced stage cancer. In particular embodiments, the subject being treated has been diagnosed with a high-grade tumor. In other embodiments, the subject being treated has been diagnosed with a low-grade tumor. In certain embodiments, the subject being treated has been diagnosed with a cancer that can metastasize. In specific embodiments, the subject being treated has been diagnosed with a metastatic cancer.

In specific embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a cervical cancer, an endometrial cancer, an ovarian cancer, a pancreatic cancer, a melanoma, a breast cancer, a colorectal cancer (e.g., a colon cancer), a bladder cancer, an astrocytic neoplasm, a glioblastoma, a pediatric Rhabdomyosarcoma, or a lung cancer (e.g., non-small cell lung carcinoma). In specific embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a melanoma, a pancreatic cancer, a breast cancer, or an ovarian cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a melanoma. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a pancreatic cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a breast cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with an ovarian cancer. In one embodiment, the subject being treated in accordance with the methods described herein has been diagnosed with a lung cancer.

In certain embodiments, the subject being treated is a hyper-progressor in response to treatment with an inhibitor of an inhibitory immune checkpoint molecule (e.g., the subject has been diagnosed with hyper-progression in response to treatment with an inhibitor of an inhibitory immune checkpoint molecule). In certain embodiments, the subject being treated is at risk of being a hyper-progressor in response to treatment with an inhibitor of an inhibitory immune checkpoint molecule (e.g., the subject has a gene amplification of M(H)DM2/4, and/or the subject has an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to its binding to the surface of intact normal cells or relative to its binding to the surface of intact cells of the cancer prior to administration of the inhibitor).

In certain embodiments, the subject being treated has previously undergone one or more other cancer therapies (e.g., vaccine, targeted therapy (such as small molecule targeted therapy), chemotherapy, radiotherapy, or immunotherapy (such as treatment with another monoclonal antibody)), and the subject's cancer has developed resistance to the one or more other cancer therapies. In one embodiment, the subject being treated is resistant to chemotherapy. In one embodiment, the subject being treated is resistant to radiotherapy. In one embodiment, the subject being treated is resistant to a small molecule targeted therapy. In one embodiment, the subject being treated is resistant to treatment with another monoclonal antibody.

In certain embodiments, the subject being treated has a type of a cancer that is known or expected to have M(H)DM2/4 (e.g., HDM2) on the surface of its cells. In specific embodiments, the subject being treated has a type of cancer, the cells of which express one or more of splice variants of HDM2 on their cell surface, for example (and without limitation), one or more of the following splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C(SEQ ID NO: 11), MDM2-D (SEQ ID NO:12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16) or MDM-KB2 (SEQ ID NO: 17).

In certain embodiments, the subject being treated has a cancer that has been tested and determined (using any assay known in the art) to carry M(H)DM2/4 (e.g., HDM2) on the plasma membrane of its cells. In particular embodiments, the subject being treated has a cancer, the cells of which have been tested and determined (by any method known in the art) to expose on their plasma membrane surface an extracellular region of HDM2 that can be targeted by an anti-HDM2 antibody or fragment (and such antibody can then be administered to the subject). In specific embodiments, the subject being treated has a cancer, the cells of which have been tested and determined (by any method known in the art) to express one or more of splice variants of HDM2 on their cell surface, for example (and without limitation), one or more of the following splice variants: MDM2-A (SEQ ID NO: 8), MDM2-A1 (SEQ ID NO: 9), MDM2-B (SEQ ID NO: 10), MDM2-C(SEQ ID NO: 11), MDM2-D (SEQ ID NO:12), MDM2-E (SEQ ID NO: 13), MDM2-F (SEQ ID NO: 14), MDM2-G (SEQ ID NO: 15), MDM2-11 (SEQ ID NO: 16) or MDM-KB2 (SEQ ID NO: 17).

7.9 Combination Therapies and Kits

In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with one or more anti-cancer therapies different from said antibody or fragment, e.g., a chemotherapy, a surgery, a radiation therapy, another antibody with an anti-cancer activity, a cytokine, a T cell therapy, a vaccine (e.g., a cellular vaccine), a small molecule with an anti-cancer activity, an anti-hormonal agent, or any other anti-cancer therapy known in the art.

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with chemotherapy. Examples of types of chemotherapeutic agents that can be used in the methods described herein include, without limitation, an alkylating agent, a nitrosourea agent, an antimetabolite, a topoisomerase inhibitor, an aromatase inhibitor, an antitumor antibiotic, an alkaloid derived from a plant, a hormone antagonist, a P-glycoprotein inhibitor, and a platimum complex derivative. Specific examples of chemotherapeutic drugs that can be used in the methods described herein include, without limitation, taxol, paclitaxel, nab-paclitaxel, 5-fluorouracil (5-FU), gemcitabine, doxorubicin, daunorubicin, colchicin, mitoxantrone, tamoxifen, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, uramustine, mustargen, ifosamide, bendamustine, carmustine, lomustine, semustine, fotemustine, streptozocin, thiotepa, mitomycin, diaziquone, tetrazine, altretamine, dacarbazine, mitozolomide, temozolomide, procarbazine, hexamethylmelamine, altretamine, hexalen, trofosfamide, estramustine, treosulfan, mannosulfan, triaziquone, carboquone, nimustine, ranimustine, azathioprine, sulfanilamide, fluoropyrimidine, thiopurine, thioguanine, mercaptopurine, cladribine, capecitabine, pemetrexed, fludarabine, methotrexate, hydroxyurea, nelarabine or clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, thioquanine, azacitidine, cladribine, pentostatin, mercaptopurine, imatinib, dactinomycin, cerubidine, bleomycin, actinomycin, luteomycin, epirubicin, idarubicin, plicamycin, vincristin, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, periwinkle, vinca, taxane, irinotecan, topotecan, camptothecin, teniposide, pirarubicin, novobiocin, merbarone, aclarubicin, amsacrine, antiandrogen, anti-estrogen, bicalutamide, medroxyprogesterone, fluoxymesterone, diethylstilbestrol, estrace, octreotide, megestrol, raloxifene, toremifene, fulvestrant, prednisone, flutamide, leuprolide, goserelin, aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, androstene, resveratrol, myosmine, catechin, apigenin eriodictyol isoliquiritigenin, mangostin, amiodarone, azithromycin, captopril, clarithromycin, cyclosporine, piperine, quercetine, quinidine, quinine, reserpine, ritonavir, tariquidar, verapamil, cisplatin, carboplatin, oxaliplatin, transplatin, nedaplatin, satraplatin, triplatin and carboplatin. In specific embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with one or more of the following chemotherapeutic agents: gemcitabine, nab-paclitaxel, capecitabine, irinotecan, and celecoxib. In specific embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with one or more of the following chemotherapeutic agents: gemcitabine, nab-paclitaxel, cisplatin, 5-FU, and paclitaxel (e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®). In specific embodiments, the cancer treated using a combination therapy described herein is a pancreatic cancer, a breast cancer, a lung cancer or an ovarian cancer.

In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine (e.g., for treatment of a non-small cell lung cancer, a pancreatic cancer or an ovarian cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with capecitabine. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with irinotecan. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with celecoxib. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®) (e.g., for treatment of a metasatic breast cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with nab-paclitaxel. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with cisplatin. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with 5-FU (e.g., for treatment of a colorectal cancer such as a colon cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with carboplatin. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and nab-paclitaxel (e.g., for treatment of a pancreatic cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and carboplatin (e.g., for treatment of an ovarian cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®) and gemcitabine (e.g., for treatment of a breast cancer, or a pancreatic cancer such as adenocarcinoma of the pancreas or metastatic adenocarcinoma of the pancreas). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and cisplatin (e.g., for treatment of anon-small cell lung cancer). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with gemcitabine and 5-FU. In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with paclitaxel (e.g., paclitaxel formulated as albumin-bound particles such as ABRAXANE®) and carboplatin (e.g., for treatment of a non-small cell lung cancer).

In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose of 1,500 mg/m². In certain embodiments, wherein the subject is human, nab-paclitaxel is administered in a dose of 300 mg/m². In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose of 1,000 mg/m². In certain embodiments, wherein the subject is human, nab-paclitaxel is administered in a dose of 125 mg/m².

In certain embodiments, the gemcitabine and/or nab-paclitaxel are administered in doses that are lower than doses used when gemcitabine and/or nab-paclitaxel are administered not in combination with an anti-cancer antibody (such as an anti-M(H)DM2/4 antibody or fragment described herein). In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose that is less than 1,500 mg/m², and/or nab-paclitaxel is administered in a dose that is less than 300 mg/m². In certain embodiments, wherein the subject is human, gemcitabine is administered in a dose that is less than 1,000 mg/m², and/or nab-paclitaxel is administered in a dose that is less than 125 mg/m². In one embodiment, wherein the subject is human, gemcitabine is administered in a dose that is equal to or less than 500 mg/m², 400 mg/m², 300 mg/m² or 200 mg/m², and/or the nab-paclitaxel is administered in a dose that is equal to or less than 62.5 mg/m², 50 mg/m², 40 mg/m², 30 mg/m², or 20 mg/m². In one embodiment, wherein the subject is human, gemcitabine is administered in a dose that is equal to or less than 900 mg/m², 800 mg/m², 700 mg/m² or 600 mg/m², and/or the nab-paclitaxel is administered in a dose that is equal to or less than 110 mg/m², 100 mg/m², 90 mg/m², 80 mg/m², or 70 mg/m². In certain embodiments, gemcitabine and/or nab-paclitaxel are administered with a frequency of every 2 weeks or less (e.g., every 3 weeks, every 4 weeks, every 6 weeks, or every 8 weeks, or less). In certain embodiments, gemcitabine is administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week. In certain embodiments, nab-paclitaxel is administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week. In certain embodiments, gemcitabine and nab-paclitaxel are administered with a frequency of once a day, 4 times per week, 3 times per week, 2 times per week, or once per week. In one embodiment, gemcitabine and/or nab-paclitaxel is administered once a week. In certain embodiments, the total duration of treatment with gemcitabine and/or nab-paclitaxel is, or is more than, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.

In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject with a cancer in combination with the chemotherapy drug(s) indicated for said cancer, which chemotherapy drug(s) can be optionally administered in the dosage and/or regime of administration indicated for said cancer. Non-limiting examples of chemotherapy drugs as well as their dosage and regime of administration indicated for various cancers are provided below.

Ovarian Cancer: The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017). Gemcitabine (Gemzar®) in combination with carboplatin is indicated for the treatment of patients with advanced ovarian cancer that has relapsed at least 6 months after completion of platinum-based therapy. The recommended dose of Gemzar® is 1000 mg/m² as an intravenous infusion over 30 minutes on Days 1 and 8 of each 21-day cycle, in combination with carboplatin (AUC 4) intravenously after Gemzar® administration on Day 1 of each 21-day cycle.

Breast Cancer: The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017). Gemcitabine (Gemzar®) in combination with paclitaxel is indicated for the first-line treatment of patients with metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless anthracyclines were clinically contraindicated. The recommended dose of Gemzar® is 1250 mg/m² intravenously over 30 minutes on Days 1 and 8 of each 21-day cycle that includes paclitaxel. Paclitaxel can be administered at 175 mg/m2 on Day 1 as a 3 hour intravenous infusion before Gemzar® administration.

Non-Small Cell Lung Cancer: The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017). Gemcitabine (Gemzar®) is indicated in combination with cisplatin for the first-line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB), or metastatic (Stage IV) non-small cell lung cancer. Every 4-week schedule: the recommended dose of Gemzar® is 1000 mg/m² intravenously over 30 minutes on Days 1, 8, and 15 in combination with cisplatin therapy; cisplatin can be administered intravenously at 100 mg/m² on Day 1 after the infusion of Gemzar®. Every 3-week schedule: the recommended dose of Gemzar® is 1250 mg/m² intravenously over 30 minutes on Days 1 and 8 in combination with cisplatin therapy; cisplatin can be administered intravenously at 100 mg/m² on Day 1 after the infusion of Gemzar®.

Pancreatic Cancer: The following information is taken from Gemzar® (gemcitabine for injection), Eli Lilly and Company, Highlights of Prescribing Information, revised March 2017, http://pi.lilly.com/us/gemzar.pdf (last accessed on Jul. 27, 2017). Gemcitabine (Gemzar®) is indicated as first-line treatment for patients with locally advanced (nonresectable Stage II or Stage III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemzar® is indicated for patients previously treated with 5-FU. The recommended dose of Gemzar® is 1000 mg/m² over 30 minutes intravenously. The recommended treatment schedule is as follows: weeks 1-8—weekly dosing for the first 7 weeks followed by one week rest; after week 8—weekly dosing on Days 1, 8, and 15 of 28-day cycles.

Metastatic Breast Cancer: The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017). ABRAXANE® is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy. Prior therapy should have included an anthracycline unless clinically contraindicated. After failure of combination chemotherapy for metastatic breast cancer or relapse within 6 months of adjuvant chemotherapy, the recommended regimen for ABRAXANE® is 260 mg/m² administered intravenously over 30 minutes every 3 weeks.

Non-Small Cell Lung Cancer: The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017). ABRAXANE® is indicated for the first-line treatment of locally advanced or metastatic non-small cell lung cancer, in combination with carboplatin, in patients who are not candidates for curative surgery or radiation therapy. The recommended dose of ABRAXANE® is 100 mg/m² administered as an intravenous infusion over 30 minutes on Days 1, 8, and 15 of each 21-day cycle. Carboplatin can be administered on Day 1 of each 21 day cycle immediately after ABRAXANE®.

Adenocarcinoma of the Pancreas: The following information is taken from ABRAXANE® (paclitaxel protein-bound particles for injectable suspension, albumin-bound), Celgene Corporation, Highlights of Prescribing Information, revised July 2015, http://www.abraxane.com/wp-content/pi/prescribing-info.html (last accessed Jul. 27, 2017). ABRAXANE® is indicated for the first-line treatment of patients with metastatic adenocarcinoma of the pancreas, in combination with gemcitabine. The recommended dose of ABRAXANE® is 125 mg/m² administered as an intravenous infusion over 30-40 minutes on Days 1, 8 and 15 of each 28-day cycle. Gemcitabine can be administered immediately after ABRAXANE® on Days 1, 8 and 15 of each 28-day cycle.

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an immunomodulator (e.g., a cytokine, an antigen, or a checkpoint targeting agent). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a checkpoint targeting agent such as, without limitation, an antagonist of PD-1, an antagonist of PD-L1, an antagonist of PD-L2, an antagonist of CTLA-4, an antagonist of TIM-3, an antagonist of GITR, an antagonist of OX40, an antagonist of LAG-3 (e.g., the antagonist of any of the above-mentioned checkpoint molecules can be an antibody, such as an inhibitory antibody to these molecules, an antibody fragment, or a small molecule). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an inhibitor of PD-1, an inhibitor of PD-L1, or an inhibitor of CTLA-4 (where the inhibitor can be an antagonistic antibody, an antibody fragment, or a small molecule).

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with radiation therapy (e.g., x-rays, gamma-rays or another source of radiation).

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with surgery (such as a surgery to remove part or all of the cancerous tumor being treated).

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a Treg-inhibitory agent.

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a T-cell therapy.

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with a tumor vaccine.

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an EGFR inhibitor (e.g., erlotinib).

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an inhibitor of one or more of: EGFR, KRAS, STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK, PI3K/AKT, TGF-β, DNMT3A, IFNγ, JAK1/JAK2/JAK3, CD274, PTEN, ART, and CDK).

In a specific embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is administered to a subject in combination with an inhibitor of p53-H(M)DM2 interaction (e.g., a small molecule inhibitor or a peptide inhibitor).

In certain embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that is not treated with a cell cycle inhibitor (i.e., the additional therapy is not an agent that inhibits cell cycle). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that is not concurrently (during the same treatment period) treated with a cell cycle inhibitor (i.e., the subject is not treated with an anti-M(H)DM2/4 antibody or fragment thereof and a cell cycle inhibitor during the same period of time, e.g., day or week). In one embodiment, an anti-M(H)DM2/4 antibody or fragment thereof described herein is used to treat a subject that has not been previously treated and is not concurrently treated with a cell cycle inhibitor.

In particular embodiments, an anti-M(H)DM2/4 antibody or fragment thereof described herein can be used before, during, or after the second therapy (e.g., a chemotherapy, a radiation therapy, a surgery, or any other therapy described herein or known in the art).

In certain embodiments, the subject being treated in accordance with the methods described herein has not received an anti-cancer therapy prior to the administration of an anti-M(H)DM2/4 antibody or fragment thereof. In other embodiments, an anti-M(H)DM2/4 antibody or fragment thereof is administered to a subject that has received an anti-cancer therapy prior to administration of the antibody or fragment. In particular, embodiments, anti-M(H)DM2/4 antibody or fragment thereof is administered to a subject recovering from or receiving an immunosuppressive therapy.

In certain embodiments, provided herein are kits comprising an anti-M(H)DM2/4 antibody or a fragment thereof, and one or more additional anti-cancer agents. In one embodiment, provided herein are kits comprising (i) an anti-M(H)DM2/4 antibody or a fragment thereof (e.g., in a therapeutically effective amount), and (ii) one or more of chemotherapeutic drugs, for example, gemcitabine, paclitaxel, or gemcitabine and nab-paclitaxel (e.g., in therapeutically effective amounts, such as any amounts described herein, which may be less than the therapeutically effective amount of the drug or drugs when the drug or drugs are used without the anti-M(H)DM2/4 antibody or fragment).

The following examples are offered by way of illustration and not by way of limitation. Various other embodiments of the invention may be practiced, given the general description provided herein.

8. EXAMPLES

The data presented herein demonstrate that specific segments (i.e., epitopes) of HDM2 are extracellularly accessible on the plasma membrane surface of intact (i.e., viable and non-permeabilized) cancer cells. Three (3) different extracellularly accessible epitopes have been identified. Specific segments of HDM2 that are extracellularly accessible include but are not limited to epitopes present in the NMC-P1, NMC-P2 and NMC-P3 peptide sequences (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively). These extracellularly accessible sequences are appropriate therapeutic and diagnostic targets for anti-HDM2 antibodies. Therefore, cancer cells expressing HDM2 on their surface membrane can be targeted with antibodies to HDM2 for diagnostic and therapeutic (i.e., anti-tumor cytotoxic and inhibitory effect) purposes.

In particular, the data presented herein demonstrated that select HDM2-specific antibodies bound to the extracellularly accessible sequences of M(H)DM2/4 on the surface membrane of intact cells of several rodent and human cancer cell lines as well as primary tumor cells from human patients. In contrast, the same HDM2-specific antibodies exhibited minimal binding to the surface membrane of normal human blood mononuclear cells. It was found that these HDM2-specific antibodies selectively bound to various cancer cells such as: intact human melanoma, uveal melanoma, pancreatic, breast, colon, lung and ovarian cancer cells in vitro. In addition, the data presented herein showed that HDM2-specific antibodies inhibited the growth of cancer cells in vitro and in vivo, strongly indicating that they can be used as therapeutic agents in vivo. Data from in vivo studies and described herein showed that select HDM2-specific antibodies inhibited tumor growth and were cytotoxic against tumors in rodent tumor models. The examples below demonstrated that select HDM2-specific antibodies were not only cytotoxic to tumor cells but also inhibited tumor growth in mouse models of pancreatic cancer, lung cancer and colon cancer. Data herein further demonstrated that only select antibodies recognized extracellularly accessible epitopes of HDM2.

Methods of Making of Antibodies Used in the Examples

Anti-HDM2 antibodies that specifically bind to (i) NMC-P1, i.e., the peptide of SEQ ID NO:1 (“NMC-100s series of monoclonal antibodies)”, (ii) NMC-P2, i.e., the peptide of SEQ ID NO:2 (“NMC-200s series of monoclonal antibodies”), and (iii) NMC-P3, i.e., the peptide of SEQ ID NO:3 (“NMC-300s series of monoclonal antibodies”) were generated using the hybridoma approach. NMC-P1, NMC-P2 and NMC-P3 peptides were conjugated to Keyhole limpet hemocyanin (KLH) using Sulfo-SMCC method (Thermo Scientific, Cat. No. 22122). Briefly, Protein-NH2 was made in Conjugation Buffer (provided by manufacturer). Twenty-fold molar excess of crosslinker was added to the protein solution and the reaction mixture was incubated for 30 minutes at room temperature. Excess cross linker was then removed using desalting column equilibrated with Conjugation Buffer. Protein-SH and desalted Protein-NH2 were then combined, mixed and incubated for 30 minutes at room temperature. The conjugation reaction was then stopped by addition of buffer containing reduced cysteine at a concentration several times greater than the sulfhydryls of Protein-SH. Following peptide conjugation, mice (BALB/c female) were immunized with the peptide NMC-P1, NMC-P2 or NMC-P3 by intraperitoneal injection at 100 μg/mouse. Boosters were injected 5-7 times to provoke immune response. Specific-antibody production was then evaluated by peptide-ELISA of the mice serum for antibody titration. Spleens of mice with high antibody titer were then harvested from each mice and single cell suspension of splenocytes were prepared. Splenocytes were then fused with SP2/0 myeloma cells (1:5 ratio). Briefly, 250 μl of EDTA was added to the mixture of splenocytes and myeloma cells. Cells were then span down and supernatant was removed. Cell pellet was then loosened and 1 mL of PEG was dispensed alongside the tube slowly over 1 min and the mixture was incubated for 5 min in 37° C. water bath. 1 mL of 100×FBS and 10 mL of IMDM medium (10% FBS) was then added and kept in the incubator for 1 hour. Cells were then centrifuged and supernatant was removed. Cell pellet was then re-suspended in IMDM (20% FBS)-containing HAT Fusion medium (hypoxanthine-aminopterin-thymidine medium). Cells were then plated in 96-well dishes and incubated for roughly 10 to 14 days. After 10-14 days when clones became visible, media supernatant from each well was tested by ELISA for its binding to their corresponding specific immunogenic peptide (NMC-P1, NMC-P2 or NMC-P3). ELISA-positive wells were then selected for further clone selection, single-cell sub-cloning and monoclonal antibody purification. Monoclonal antibody selection was done by peptide-ELISA using NMC-P1, NMC-P2 or NMC-P3 peptide antigen. To further select antibodies that react with the peptide antigen as well as with plasma membrane HDM2, binding assays were also performed for mAb selection by cell-ELISA on intact cancer cells.

Predicted Reactivities of the Generated Antibodies

Predicted reactivities of the NMC-100s series, NMC-200s series, and NMC-300s series of monoclonal antibodies with different HDM2 isoforms/variants are presented in Table 1 and Table 2. Predictions were made based on whether the respective isoform contains the sequence corresponding to the sequence of P1, P2 or P3 (and knowledge of to which peptide (P1, P2, or P3) the respective NMC series mAb binds, based on which peptide was used as immunogen for which series).

TABLE 1 Predicted reactivities of the NMC-100s series, NMC-200s series, and NMC-300s series of monoclonal antibodies with different HDM2 isoforms/variants. Reactivity Reactivity Reactivity Uniprot with mAb with mAb with mAb Accession Amino acid residues of HDM2 (SEQ ID NMC-100s NMC-200s NMC-300s # Isoform NO: 4) that are missing in the isoform series series series Q00987-2 Mdm2-A 28-222 + + + Q00987-3 Mdm2-A1  28-222; + + + 275-300  Q00987-4 Mdm2-B 28-300 + + + Q00987-5 Mdm2-C 53-222 + + + Q00987-6 Mdm2-D 30-388 + + + Q00987-7 Mdm2-E  76-102: + + − YCSNDLLGDLFGVPSFSVKEHRKIYTM → NDCANLFPLVDLSIRELYISNYITLGI; 103-491  Q00987-9 Mdm2-F 53-97  + + + Q00987-10 Mdm2-G 115-169  + + + Q00987-11 Isoform 11 1-1: M → MVRSRQM + + + MDM2- 157-248  + + + KB2 Q00987-8 Mdm2- 1-61 − − + alpha Q00987 Canonical  1-491 + + +

TABLE 2 Predicted reactivities of the NMC-100s series, NMC-200s series, and NMC-300s series of monoclonal antibodies with different HDM2 isoforms/variants. Reactivity Reactivity Reactivity with mAb with mAb with mAb NMC-100 NMC-200 NMC-300 NCBI Accession # series series series EAW97204.1 + + − EAW97206.1 + + + EAW97213.1 + + + EAW97210.1 + + + EAW97201.1 + + − EAW97202.1 + + + EAW97203.1 + + + EAW97212.1 + + + EAW97209.1 + + + EAW97205.1 + + + AFM80534.1 − − + CAP16722.1 + + − CAP16731.1 + + − AAA75518.1 + + − CAD36961.1 + + − CAP16717.1 + + − AFM80529.1 + + + AFM80530.1 + + + CAC07811.1 + + + AAA75517.1 + + + AFM80531.1 + + + CAP16743.1 + + − CAC07810.1 + − + CAP16730.1 + + − AAL13247.1 + + + AFM80533.1 + + + CAD36959.1 + + − CAP16712.1 + + − CAD23252.1 + + + CAP16721.1 + + − CAP16716.1 + + − AFM80535.1 + + + CAP16728.1 + + − CAP16734.1 + + − AAL13243.1 + + − AFM80536.1 + + + AFM80537.1 + + + AAA75515.1 + + + AFM80538.1 + + − AFM80539.1 + + + CAC07809.1 + + + CAD23251.1 + + + NP_001138812.1 + + + AAA75514.1 + + + CAP16729.1 + + − CAP16733.1 + + − AFM80540.1 + + + XP_006719463.1 − − + CAP16735.1 + + − CAP16708.1 + + + XP_006719462.1 − − + AAL40179.1 + + + NP_001138811.1 + + + NP_001138809.1 + + + AAL40180.1 + + + CAP16705.1 + + + ACX31156.1 + + + CAP16732.1 + + + CAP16703.1 + + − CAP16738.1 + + − XP_005268929.1 + + + NP_002383.2 + + + CAD36962.1 + + − CAP16704.1 + + + CAP16715.1 + + − CA1D79459.1 + + − CAP16707.1 + + − AAL13242.1 + + − CAP16718.1 + + − CAP16727.1 + + − CAD79456.1 + + − AAL13244.1 + + − CAP16739.1 + + − CAD79457.1 + + − CAD36960.1 + + − CAD79458.1 + + − CAP16725.1 + + − CAP16719.1 + + − CAP16740.1 + + − CAP16736.1 + + − AFM80527.1 + + − AAL13245.1 + + − CAP16737.1 + + − AFM80528.1 + + + AAL13246.1 + + + CAC07812.1 + + + AAF42995.1 − + +

Antibodies Used in the Examples

Monoclonal antibody NMC-103 is an antibody that binds to NMC-P1 (SEQ ID NO:1) (it is one of the NMC-100s series of antibodies). Monoclonal antibody NMC-204 is an antibody that binds to NMC-P2 (SEQ ID NO:2) (it is one of the NMC-200s series of antibodies). Monoclonal antibody NMC-303 is an antibody that binds to NMC-P3 (SEQ ID NO:3) (it is one of the NMC-300s series of antibodies). The heavy chain/light chain frame work region sequences, complementarity determining region (CDR) sequences, and variable region sequences of these antibodies are listed in Section 8, below.

The following anti-HDM2 antibodies were used in the experiments described in Examples 1-9: (i) purified NMC-103 mouse monoclonal antibody (mAb) of the IgG1 isotype (NMC-103 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns), (ii) purified NMC-204 mouse mAb of the IgG3 isotype (NMC-204 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns); (iii) purified NMC-303 mouse mAb of the IgM isotype (NMC-303 mAbs produced by single-cell cloned hybridoma cells were purified on protein G/A columns); (iv) an anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01); (v) an anti-HDM2 antibody termed “MDM2 Antibody (D-7)” (Santa Cruz, Cat. No. sc-13161); (vi) an anti-HDM2 antibody termed “p-MDM2 Antibody (2G2)” (Santa Cruz, Cat. No. sc-53368); (vii) an anti-HDM2 antibody termed “MDM2 Antibody (SPM344)” (Santa Cruz, Cat. No. sc-56430); (viii) an anti-HDM2 antibody termed “MDM2 Antibody (SMP14)” (Santa Cruz, Cat. No. sc-965); (ix) an anti-HDM2 antibody termed “Anti-MDM2 clone 4B2C1.11” (EMD Millipore, Cat. No. MABE331); (x) an anti-HDM2 antibody termed “Anti-MDM2 clone 3G9” (EMD Millipore, Cat. No. 04-1530); (xi) an anti-HDM2 antibody termed “Anti-MDM2 clone 2A10” (EMD Millipore, Cat. No. MABE281); (xii) an anti-HDM2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” (EMD Millipore, Cat. No. OP46); (xiii) an anti-HDM2 antibody termed “Anti-MDM2 (Ab-3) Mouse mAb (4B11)” (EMD Millipore, Cat. No. OP143); (xiv) an anti-HDM2 antibody termed “Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)” (EMD Millipore, Cat. No. OP144); (xv) an anti-HDM2 antibody termed “Anti-MDM2 (Ab-5) Mouse mAb (4B2C1.11)” (EMD Millipore, Cat. No. OP145); (xvi) an anti-HDM2 antibody termed “MDM2 Antibody (C-18): sc-812” (Santa Cruz, polyclonal, Cat No. sc-812); and (xvii) n anti-HDM2 antibody termed “MDM2 Antibody (N-20): sc-813” (Santa Cruz, polyclonal, Cat No. sc-813).

8.1 Example 1: Selection of HDM2-Specific mAbs to Extracellularly Accessible Epitopes of HDM2

Utilizing enzyme-linked immunosorbent assay (ELISA) the data presented herein showed that mAb NMC-103 specifically bound to peptide NMC-P1 (SEQ ID NO:1) corresponding to amino acids 1-15 of HDM2, while mAb NMC-204 specifically bound to NMC-P2 peptide (SEQ ID NO:2) corresponding to amino acids 15-25 of HDM2, and mAb NMC-303 specifically bound to NMC-P3 peptide (SEQ ID NO:3) corresponding to amino acids 475-491 of HDM2. Moreover, immunoblot analysis presented here shows that mAbs NMC-103, NMC-204 and NMC-303 recognized the full-length recombinant HDM2 protein.

Peptide-ELISA Methodology: 5 μg/ml of NMC-P1, NMC-P2 or NMC-P3 peptide antigen was dried onto a 96-well ELISA plate overnight. Plates were then blocked with 5% BSA in 1× phosphate-buffered saline (PBS) (100 μl/well) for 2 hours at room temperature. Microplate wells were then washed 5 times with 300 μl of ice cold 1×PBS. Appropriate dilutions of monoclonal antibodies NMC-103, NMC-204 or NMC-303 in 1% bovine serum albumin (BSA) in PBS solution were then incubated with their corresponding peptides (NMC-P1, NMC-P2 or NMC-P3, respectively) at room temperature. After 2 hours, wells were washed 5 times with 300 μl ice-cold PBS/well and 100 μl secondary antibody (HRP-labeled Goat anti-Mouse F(ab′)2 H&L cross-adsorbed secondary antibody (HRP-GaM F(ab′)2), ThermoFisher, Cat. No. A24524) diluted 1:2500 to 1:5000 in PBS-1% BSA solution were added and the incubation continued at room temperature. After 1 hour, the wells were washed again 5 times with 200 μl of ice-cold PBS and 50 μL of the TMB Substrate Solution (1-Step™ Ultra TMB-ELISA, ThermoFisher, Cat. No. 34028) were added to each well and color was allowed to develop for 30 minutes at room temperature. The reaction was stopped by addition of 50 μL of stop solution (ThermoFisher, Cat. No. SS04) to each well and absorbance of each well was read immediately for optical density (OD) at 450 nm.

Competition assays: As for peptide antigen binding competition experiments, 1 μl/ml of mAb NMC-103 was pre-incubated with 10 μl/ml of NMC-P1, NMC-P2 or NMC-P3 for 1 hour at room temperature. MAb NMC-103 was then incubated with ELISA plates coated with NMC-P1 as described above.

Western Blot: As for immunoblots, 1 μg/well of recombinant HDM2 protein (GST tagged, Abcam, Cat. No. ab188727) was separated on 8-16% sodium dodecyl sulfate (SDS) polyacrylamide gel and proteins were then transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% milk followed by incubation with monoclonal antibody NMC-103, NMC-204 or NMC-303 at 1 μg/ml in 1% milk in PBS for 2 hours. The membrane was then washed ×3 times (10 minutes each) and incubated for 1 hour with corresponding HRP-conjugated secondary antibody (Goat anti-Mouse IgG (H&L), F(ab′)2 Frag Cross-adsorbed HRP (HRP-GaM F(ab′)2), ThermoFisher, Cat. No. A24524) diluted 1:5000 in 1% BSA-PBS. The membrane was then washed ×3 times and incubated with Pierce ECL Plus Western Blotting Substrate (ThermoFisher, Cat. No. #32132) for 10 min before developing on a LICOR Scanner.

The peptide-ELISA experiments shown in FIG. 1 demonstrated selective and specific binding of monoclonal antibodies (mAb) NMC-103, NMC-204 and NMC-303 to their corresponding peptide antigens. FIG. 1A shows that NMC-103 bound to NMC-P1 peptide while NMC-204 did not bind to NMC-P1. FIG. 1B shows that mAb NMC-204 bound to NMC-P2 peptide while mAb NMC-103 did not bind to NMC-P2. FIG. 1C shows that mAb NMC-303 bound to NMC-P3 peptide while NMC-204 did not bind to NMC-P3. Thus, the Peptide-ELISA results demonstrated that each of the mAbs NMC-103, NMC-204 and NMC-303 selectively bound to their corresponding antigen peptides on the ELISA-plate and not to other peptides. FIG. 1D shows that while mAb NMC-103 bound to NMC-P1, pre-incubation of NMC-P1 peptide with mAb NMC-103 abolished the binding of NMC-103 to NMC-P1 peptide on the plate. In contrast, pre-incubation of mAb NMC-103 with either NMC-P2 or NMC-P3 did not affect the binding of mAb NMC-103 to NMC-P1. These experiments demonstrate the specificity of mAbs NMC-103, NMC-204 and NMC-303 for their corresponding peptide antigens.

The results of the immunoblot experiments in FIG. 2 demonstrated that mAbs NMC-103, NMC-204 and NMC-303 reacted with the recombinant full-length HDM2 protein. The recombinant protein has a GST tag at its N-terminal which brings the molecular weight to approximately 83 kD. Lane 2 of the immunoblot shows the reactivity of a commercially available antibody raised against amino acids 100-320 of MDM2 of human origin (MDM2 (D-7); Santa Cruz, Cat. No. sc-13161) to the full-length recombinant HDM-2 protein. Lane 3 of the immunoblot shows the reactivity of mAb NMC-103 with a single band at approximately 83 kD, corresponding to the recombinant HDM2-GST protein. Lane 4 of the immunoblot shows the binding of mAb NMC-204 to a single band at approximately 83 kD, corresponding to the recombinant HDM2-GST protein. Lane 5 of the immunoblot shows reactivity of mAb NMC-303 with the recombinant HDM2-GST protein at 83 kD. In contrast, in Lane 1, control mouse IgG (Abcam, Cat. No. ab18447) did not react with the recombinant HDM2-GST protein. The data presented here further demonstrated that mAbs NMC-103, NMC-204 and NMC-303 recognized full-length HDM2.

8.2 Example 2: HDM2-Specific Antibodies Bound to Extracellularly Accessible Epitopes of M(H)DM2/4 on Intact Cancer Cells from Different Rodent and Human Cancer Cell Lines and Freshly Isolated Primary Human Cancer Cells but not to Normal Cells

General description: Utilizing ELISA, the data presented herein demonstrated the binding of mAbs NMC-103, NMC-204 and NMC-303 to extracellularly accessible epitopes of M(H)DM2/4 within the NMC-P1, NMC-P2 and NMC-P3 sequences, respectively, on the plasma membrane of human breast cancer cells, human triple negative breast cancer cells, human melanoma cells, chemo-resistant human ovarian cancer cells as well as primary patient-derived human ovarian cancer cells, human and mouse pancreatic cancer cells, mouse colon cancer cells and mouse lung cancer cells. Data presented herein also demonstrated the selective binding of these mAb to cancer cells and not to normal untransformed cells. Moreover, peptide-antigen competition results showed that each of these mAbs specifically bound to their corresponding NMC-P1, NMC-P2 or NMC-P3 regions of the plasma membrane M(H)DM2/4.

Cell-ELISA Methodology: 8,000-10,000 cells/well of a 96-well microplate were grown overnight. The next day, unbound cells were washed off with sterile 1×PBS. The cells in each well were fixed with freshly prepared 4% buffered paraformaldehyde (pH 7.2) for 1 hour followed by 3 washes with 1×PBS. The wells were then blocked with 5% BSA in PBS (100 μl/well) for 2 hours at room temperature. Microplate wells were then washed 5 times with 300 μl of ice cold 1×PBS. MAbs NMC-103, NMC-204 or NMC-303 at 1 μg/mL in 1% BSA/PBS were then incubated with various cancer or normal untransformed cells for 2 hours at room temperature. Wells were then washed with 300 μl of ice-cold 1×PBS for 5 times and corresponding secondary antibody HRP-GaM F(ab′)2 diluted 1:2500 or 1:5000 in PBS with 1% BSA were added at 100 μl/well for 1 hour at room temperature followed by washing 5 times with 300 μl of ice-cold 1×PBS. TMB Substrate Solution (1-Step™ Ultra TMB-ELISA, ThermoFisher, Cat. No. 34028) was then added at 50 μL to each microplate well and incubated at room temperature for 30 minutes. The reaction was stopped by addition of 50 μL of stop solution (ThermoFisher, Cat. No. SS04) to each well and absorbance of each well was measured at OD450 nm. The absorbance value of each experimental well was corrected for the absorbance value obtained from wells treated with isotype-identical mAbs included in each experiment. The results are thus expressed as “relative binding”.

FIGS. 3-5 show the relative binding of mAbs NMC-103, NMC-204 and NMC-303 to intact cells of different types of human (A) and rodent (B) cancers. FIG. 3 shows reactivity of mAb NMC-103 to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells that are resistant to adriamycin, melphalan, and cisplatin, primary patient-derived human ovarian cancer OVCA4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells. FIG. 4 presents the reactivity of NMC-204 monoclonal antibody to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human ovarian cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells. FIG. 5 shows reactivity of NMC-303 monoclonal antibody to human breast cancer MCF-7 cells, human triple negative breast cancer HCC1806 cells, human pancreatic cancer MIA PaCa-2 cells, human ovarian cancer OVCAR-3 cells, primary patient-derived human ovarian cancer OVCA.4 cells, human melanoma A2058 cells, human uveal melanoma 92.1 cells, mouse colon cancer MC-38 cells, mouse Lewis Lung LL/2 cells and mouse pancreatic Panc02 cells.

FIG. 6 demonstrates that NMC-204 did not react with normal human PBMCs. In contrast to cancer cells, neither mAb NMC-103, NMC-204 nor NMC-303 bound to normal intact cells (the data for NMC-103 and NMC-303 is not shown). Data presented herein show that, while mAb NMC-204 reacted with human pancreatic cancer MIA PaCa-2 cells, no binding was seen above the background when mAb NMC-204 was incubated with freshly isolated normal human peripheral blood mononuclear cells (PBMCs). Moreover, FIG. 6 demonstrates that while mAb NMC-204 did not react with normal human PBMCs (FIG. 6 left; white bar graph), these cells showed strong reactivity with an mAb against CD3e, a cell surface marker for T cells (FIG. 6 left; shaded bar graph).

FIG. 7 depicts mAb NMC-103 and NMC-204 saturation curves. FIG. 7A shows that cell-ELISA binding of mAb NMC-103 to intact MIA PaCa-2 cells increased as the concentration of the antibody increased. However, this binding reached a plateau at concentrations of above 50 μg/mL of mAb NMC-103, demonstrating the saturation of antigen binding sites by the specific antibody NMC-103. Moreover, FIG. 7B depicts the binding saturation curve of mAb NMC-204 to intact MIA PaCa-2 cells. The binding of mAb NMC-204 to its antigen on the intact MIA PaCa-2 cells reached saturation at concentrations above 50 μg/mL of mAb NMC-204, demonstrating the binding saturation of antigen sites for mAb NMC-204.

8.3 Example 3: mAb NMC-103. NMC-204 and NMC-303 were Specific for Extracellularly Accessible NMC-P1. NMC-P2 and NMC-P3 Sequences of HDM2 on Intact Cancer Cells, Respectively

The data shown in FIG. 8 demonstrated the specificity of mAbs NMC-103, NMC-204 and NMC-303 for NMC-P1, NMC-P2 and NMC-P3 sequences of HDM2 that are extracellularly accessible on the membrane of cancer cells. FIG. 8A shows the binding of mAb NMC-103 to intact human pancreatic cancer MIA PaCa-2 cells (left, solid bar). The binding of monoclonal antibody NMC-103 to its epitope of HDM2 that is accessible on the surface plasma membrane of MIA PaCa-2 cells was completely abolished when mAb NMC-103 was competed with soluble NMC-P1 (middle column, solid bar). In contrast, competition with NMC-P2 did not have any effect on the reactivity of NMC-103 with the epitope of HDM2 to which it binds and which is accessible on the cell surface of the MIA PaCa-2 cells (solid, filled bar).

On the other hand, as shown in FIG. 8B, mAb NMC-204's reactivity with intact MIA PaCa-2 cells was competed with NMC-P2 peptide and not with NMC-P1, demonstrating epitope specificity for another extracellularly accessible epitope/peptide of HDM2 expressed on the plasma membrane of MIA PaCa-2 cells.

The observation of the specificity of the newly generated mAbs for certain epitopes on HDM2 was further extended by the observation that mAb NMC-303's binding to intact MIA PaCa-2 was competed with NMC-P3 peptide and not with NMC-P2 (FIG. 8C). Taken together, these data demonstrate the specificity of mAbs NMC-103, NMC-204 and NMC-303 for 3 different extracellularly accessible sequences of HDM2, namely NMC-P1, NMC-P2 and NMC-P3, respectively.

As demonstrated in FIG. 9, when mAb NMC-103 was pre-incubated with full-length recombinant HDM2, the binding of the antibody to intact MIA PaCa-2 cells was reduced. This result further confirms the specific binding of NMC antibodies to HDM2.

8.4 Example 4: A Plasma Membrane Marker Antibody, Antibody NMC-103 and Antibody NMC-204 Stain Plasma Membrane Surface of Intact Cells, while an Intracellular Marker Antibody does not

The data presented herein (FIG. 10) showed that select HDM2-specific antibodies bound to the extracellularly accessible HDM2 on the plasma membrane of intact cancer cells (human pancreatic MiaPaCa-2 cells) that were treated with EDTA (10 mmols; pH7.2; 5 min, 37° C.). EDTA treatment therefore, appeared to have little or no effect on the expression of HDM2 on the cancer cells' surface membrane, strongly suggesting that the HDM2 protein antigen is an integral membrane protein. Additional data presented here show that antibodies to plasma membrane markers such as E-Cadherin (Transduction Lab, Cat. No. C37020) bound to intact cells, while antibodies specific for markers that are located intracellularly such as Cytochrome-C(Santa Cruz, Cat. No. sc-13156) were unable to bind to their targets due to the inaccessibility of the intracellular compartments in the intact cells.

8.5 Example 5: Extracellular Protease Digestion of Intact Cancer Cells Removed Extracellularly Accessible Sequences of M(H)DM2 on the Plasma Membrane of Intact Cancer Cells

MAbs NMC-103, NMC-204 and NMC-303 were raised against amino acids 1-15 (NMC-P1), 15-25 (NMC-P2) and 475-491 (NMC-P3) of HDM2, respectively, and were highly specific for HDM2. As shown in the immunoblot of FIG. 2, all three mAbs NMC-103, NMC-204 and NMC-303 recognized and bound to purified HDM2 protein, providing evidence that the unique antigenic epitopes recognized by the three mAbs in NMC-P1, NMC-P2 and NMC-P3, respectively, are de facto structures of intact HDM2. The results of Cell-ELISAs in FIGS. 3-5 and 8 took the observations one step further by providing evidence that the M(H)DM2 epitopes are expressed extracellularly on the surface membrane of intact rodent and human cancer cells: (1) mAbs NMC-103, NMC-204 and NMC-303 bound to intact cancer cells (FIG. 3-5); (2) each mAb's binding was effectively competed with by its specific peptide but not by the other peptides (FIG. 8); (3) NMC-mAb's binding was also effectively competed by the intact HDM2-protein (FIG. 9); (4) virtually no binding could be seen of mAbs NMC-103, NMC-204 or NMC-303 to intact normal human PBMCs (FIG. 6).

To further solidify the finding of the expression of M(H)DM2 on the surface membrane of cancer cells, binding by indirect immunofluorescence of mAbs NMC-103, NMC-204 and NMC-303, respectively, to cancer cells was examined before and after protease treatment of the cells. Extracellular protease digestion has been validated as a powerful means for evaluation of a cell surface localization of plasma membrane proteins (Besingi and Clark, 2015, Nat. Protoc. 10(12): 2074-2080; Schülein et al., 1996, J. Biol. Chem. 271(46):28844-28852): loss of binding of a cell surface antigen-specific antibody to cells after protease treatment indicates not only that the antigen is accessible to proteolysis but also, and most importantly, that the antigen is exposed on the extracellular surface of the cells. The accessibility, therefore, of extracellularly displayed M(H)DM2 protein antigen and particularly of the amino acid sequences of NMC-P1, NMC-P2 and NMC-P3, was evaluated by the antigen's susceptibility to digestion by trypsin. The effect of trypsin on the binding of mAbs NMC-103, NMC-204 and NMC-303, was then examined using cell ELISA and HRP reactivity at OD450 nm as described above.

Flow Cytometry Methodology: Cells that had been allowed to grow to about 80% confluency in 25 cm² tissue culture flasks were released with either EDTA (10 mmols; pH7.2; 5 min, 37° C.) or Trypsin (Gibco TrypLE Express), fixed with freshly made 4% buffered (pH 7.4) paraformaldehyde for 1 h at room temperature, washed several times with a large volume of ice-cold PBS, and blocked for 30 minutes at room temperature with 5% human serum albumin in PBS (intact, non-permeabilized cells). After establishing viability, the cell number was adjusted to 10⁶ cells/ml and kept in ice-cold PBS until staining. Another set of the same cells was released with either EDTA or Trypsin and fixed with 4% buffered paraformaldehyde as above, then washed and blocked with 5% human serum albumin in PBS, and was treated separately with Triton X-100 (0.1% in PBS) (International Biotechnologies Inc. 07100) for 5 minutes at room temperature for membrane permeabilization. After washing off the Triton X-100 solution with PBS the cells were adjusted to 10⁶ cells/ml and stored in ice-cold PBS. Cells (10⁶/ml) from each preparation were then incubated either with 5 μg/ml of mAb to Na+/K+ ATPase α-1 (Abcam, Cat. No. ab2826), 5 μg/ml of mAb NMC-103, or 5 μg/ml of mAb NMC-204 for 90 min at room temperature. Following primary antibody incubation, cells were washed 3 times with ice-cold PBS followed by incubation with rabbit anti-mouse PE-Cy5 labeled secondary antibody (PE-Cy5-RaM IgG (H&L); Invitrogen, Cat. No. M35018) at room temperature. Sixty minutes later the cells were washed 3 times with PBS and subjected to flow cytometry analysis (BD Canto Flow Cytometry). Controls to establish background fluorescence included the analysis of samples of unstained cells as well as cells that had been processed with secondary antibody under the same conditions as used in the experimental samples.

To evaluate the extracellular versus intracellular accessibility of antibodies on an intact cell, the binding of an antibody to a plasma membrane marker, E-Cadherin (Transduction Lab C37020) and the binding of an antibody to an intracellular protein, Cytochrome-C(santa cruz sc-13156) were tested using intact human pancreatic cancer MiaPaCa-2 cells. The cell-ELISA results in FIG. 10 demonstrate the binding of E-Cadherin antibody to intact, EDTA-released MiaPaCa-2 cells (first bar from left). In contrast, mAb to Cytochrome-C showed only minimal binding to EDTA-released cells (second bar from left), which is indicative of the inaccessibility of intracellular targets in intact cells to large molecules such as antibodies. On the other hand, both mAbs NMC-103 and NMC-204 bound to intact, EDTA-released MIAPaCa-2 cells, establishing strong evidence for their interaction with their epitopes present in the extracellularly accessible sequences of HDM2.

To further validate that the epitopes of the plasma membrane M(H)DM2 are in fact extracellularly accessible sequences on cancer cells, binding of mAbs NMC-103, NMC-204 and NMC-303 was performed on mouse Lewis Lung LL/2 cancer cells under treatment conditions of EDTA versus trypsin. Both of these conditions were also tested on intact versus permeabilized cells. FIG. 11 panels A, B and C present flow cytometry data on % cells stained with mAbs NMC-103, NMC-204 and anti-Na+/K+ ATPase α-1, respectively. Each antibody was reacted with cells from four treatment conditions: EDTA-treated intact cells, EDTA-treated permeabilized cells, trypsin-treated intact cells, trypsin-treated permeabilized cells. EDTA-treated intact cells showed staining with mAb NMC-103 (70.4%), NMC-204 (51.2%) and anti-Na+/K+ ATPase α-1 antibody (29.9%). When EDTA-treated cells were permeabilized, staining with NMC-103, NMC-204 and anti-Na+/K+ ATPase α-1 increased to 75.8%, 52.85 and 63.8%, respectively. However, when compared with EDTA-treated cells, the binding of mAbs NMC-103, NMC-204 and anti-Na+/K+ ATPase α-1 to intact cells that were treated with trypsin reduced to 36.8%, 27% and 20.5%, respectively, due to extracellular protease digestion of antibody recognition epitopes. However, when trypsin-treaded cells were permeabilized, the above reduction in binding was compensated by reactivity of the antibodies to intracellular MDM2 or Na+/K+ ATPase α-1. Taken together, results presented herein further demonstrate the accessibility of extracellular sequences of transmembrane M(H)DM2 on intact cancer cells which can be cleaved by extracellular protease treatment.

8.6 Example 6: Specific HDM2 Antibodies Inhibited the Growth of Intact Cancer Cells In Vitro

Data presented herein demonstrate that antibodies that recognized extracellularly accessible sequences of HDM2 were able to inhibit the growth of cancer cells in vitro.

Methodology used: Human pancreatic cancer MIAPaCa-2 cells (8,000 cells/well) were pre-incubated with mAb NMC-103, NMC-204 and anti-Cytochrome-C antibody (Santa Cruz, Cat No. sc-13156) at 1, 5 and 10 μg/mL, respectively. Cells in the presence of one of the antibodies at various concentrations were then plated in triplicate in 96-well cell culture plates overnight. The next day, each plate well was analyzed morphologically and a picture was taken from each well using EVOS FL microscope. Cell count was also performed and the number of living cells was graphed as % of control.

FIG. 12 (A-D) demonstrates that human pancreatic cancer MIAPaCa-2 cells that were treated with mAb NMC-103 or NMC-204 exhibited a concentration-dependent growth inhibition, while an antibody against an intracellular marker (i.e., Cytochrome C) had no effect on the growth of these cancer cells. As demonstrated in FIG. 12A, MIAPaCa-2 cells treated with 1 μg/mL (top panel, left image), 5 μg/mL (to panel, middle image) and 10 μg/mL (top panel, left image) mAb NMC-103 showed an increasing effect in inhibition of their growth. FIG. 12B presents images of the cells treated with 1, 10 and 20 μg/mL of mAb NMC-204, showing a concentration-dependent inhibition of cell growth. In contract, FIG. 12C demonstrates that treatment of MIAPaCa-2 cells with an antibody against Cytochrome-C, which was used as an intracellular marker control, had no effect on the growth of these cells at any of the 1, 10 and 20 μg/mL concentration. FIG. 12D quantifies the growth inhibitory effect of mAb NMC-103 at 1 μg/mL (65%), 5 μg/mL (84%) and 10 μg/mL (91%) (solid black line) and of NMC-204 at 1 μg/mL (65%), 5 μg/mL (73%) and 10 μg/mL (77%) (dashed black line). In contrast, treatment with an antibody to Cytochrome C (an intracellular target; Santa Cruz, Cat. No. sc-13156) showed no sign of growth inhibition at 1 μg/mL (100%), 5 μg/mL (100%) and 10 μg/mL (104%) (solid gray line).

8.7 Example 7: HDM2-Specific Antibodies Initiated Complement-Dependent Cytotoxicity (CDC) Against Cancer Cells

The data presented herein show the ability of HDM2-specific antibodies to not only bind to the extracellularly accessible sequences of HDM2 on the surface membrane of cancer cells but also to initiate a potent cytotoxic effect in the presence of fresh normal human serum (NETS). The cytotoxic effect was measured in pancreatic cancer cells as well as in normal human fibroblasts. Data presented herein demonstrated one such cytotoxic effect when human pancreatic cancer MIAPaCa-2 cells were treated with mAb NMC-103 in the presence of NHS.

Methodology used: Cells seeded in 24-well dishes were grown overnight at 37° C. in a humidified atmosphere supplied with 5% CO₂-95% air mixture. The following day the cells were thoroughly washed with ice-cold PBS and incubated with HDM2-specific mAb NMC-103 diluted in culture medium containing 1% BSA to a final antibody concentration of 30 μg/ml. After 30 min, NHS was added to the cells at a final concentration of 1:10 in the culture medium with 1% BSA together with 1% propidium iodide (PI) solution. Cells were then incubated with NHS for 2 hours and images were taken using EVOS FL fluorescent microscope (Life Technologies).

FIG. 13 demonstrates the cytotoxic effect of HDM2-specific mAb NMC-103 against human pancreatic cells. mAb NMC-103 in the presence of NHS triggered complement-mediated cytotoxicity in cancer cells, resulting in the death of the cancer cells as evident by the nuclear uptake of the cell-death marker Propidium Iodide (PI). FIG. 13C provides a quantitative representation of HDM2-specific antibody complement-dependent-cytotoxicity (CDC) against human pancreatic cancer cells. Cells treated with mAb NMC-103 (FIG. 13B) in the presence of NHS demonstrated cytotoxicity over 2 hours post-treatment as compared with cells treated with NHS in the absence of any antibody (FIG. 13A). Control experiment was performed with anti-Cytochrome C antibody or with cells left untreated in the presence of NHS.

8.8 Example 8: Evaluation of Other Anti-HDM2 mAb Antibodies in their Binding to NMC-P1, NMC-P2 and NMC-P3 and to Intact Cancer Cells

There are a number of HDM2-specific monoclonal antibodies that are commercially available. To evaluate the binding of commercially available mAbs to HDM2, utilizing peptide- and cell-ELISA, we tested a number of such mAb for their binding to intact cancer cells. These mAbs included antibodies that were raised against various regions from the N-terminus or C-terminus, or segments in the middle of the HDM2 protein, and were tested for their binding to newly identified extracellularly accessible NMC-P1 and NMC-P2 sequences as well as to intact cancer cells.

Table 3 summarizes the commercially available mAbs that were tested, the companies that generated these mAbs, and amino acid residues of HDM2 against which they had been raised.

TABLE 3 Commercially available monoclonal antibodies that were tested. Antigen Residues Antibody Company Cat. No. on HDM2 MDM2 monoclonal ABNOVA H00004193- 101-200 antobpdy (MO1), clone M01 1A7 MDM2 Antibody (D-7) SANTA CRUZ SC-13161 100-320 p-MDM2 Antibody (2G2) SANTA CRUZ SC-53368 180-190 MDM2 Antibody SANTA CRUZ SC-56430 154-167 (SPM344) MDM2 Antibody (SMP14) SANTA CRUZ SC-965 154-167 Anti-MDM2 clone MILLIPORE MABE331 Recombinant 4B2C1.11 human MDM2 Anti-MDM2 clone 3G9 MILLIPORE 04-1530 Anti-MDM2 clone 2A10 MILLIPORE MABE281 His-tagged recombinant human MDM2 Anit-MDM2 (Ab-1) Mouse CALBIOCHEM OP46 26-169 mAb (IF2) Anti-MDM2 (Ab-3) Mouse CALBIOCHEM OP143 383-491 mAb (4B11) Anti-MDM2 (Ab-4) Mouse CALBIOCHEM OP144 153-222 mAb (2A9C1.18) Anti-MDM2 (Ab-5) Mouse CALBIOCHEM OP145 19-50 mAb (4B2C1.11)

Utilizing Peptide-ELISA, FIGS. 14A and 14B demonstrated the lack of binding of any of these mAbs to either NMC-P1 or NMC-P2. In contrast, NMC-103 and NMC-204 showed strong binding to NMC-P1 and NMC-P2, respectively.

In these experiments, it was further evaluated whether HDM-2 binding components such as peptides, for example PNC-27, interfered with the binding of mAb antibodies to NMC-P1 and NMC-P2. Even though it is not known where exactly the HDM2-binding component of PNC-27 and PNC-28 peptides binds on HDM2, it has been reported to bind within amino acids 25-109 of HDM2 (Do et al., 2003, Oncogene 22(10):1431-1444 (“Do 2003”); Chène, 2003, Nat. Rev. Cancer 3(2):102-109). To rule out that PNC-27 and PNC-28 peptides bind to NMC-P1 or NMC-P2, the peptides' binding to these HDM2 sequences was evaluated by peptide-ELISA. As shown in FIGS. 14A and 14B, PNC-27 (10 μg/mL) did not compete with the binding of NMC-103 and NMC-204 (1 μg/mL) to NMC-P1 and NMC-P2, respectively. It was found that PNC-27 does not bind to NMC-P1 or NMC-P2 regions of HDM2). Overall, these observations demonstrate that the HDM2-binding component of PNC-27 does not react with NMC-P1 or NMC-P2. Moreover, it has been demonstrated that the HDM-2 binding components of PNC-27 and PNC-28 have no anti-cancer activity by themselves and are only active when attached to a membrane resident peptide (MRP or Penetratin sequence) (see Kanovsky 2001, Do 2003, and Bowne 2008).

In the analysis of mAbs by cell-ELISA, despite the fact that none of the tested commercially available antibodies bound to either NMC-P1 or NMC-P2, an anti-HDM2 antibody raised against amino acid residues 19-50 of HDM2, termed “Anti-MDM2 (Ab-5) Mouse mAb (4B2C1.11)” (EMD Millipore, Cat. No. OP145), and an anti-HDM2 antibody termed “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) were identified as reactive with intact cancer cells (see FIG. 15A, showing data for MDM2 monoclonal antibody (M01), clone 1A7). MDM2 monoclonal antibody (M01), clone 1A7 was raised against amino acid 101 to 200 of full-length HDM2. Thus, the data demonstrate that epitopes of HDM2 other than NMC-P1, NMC-P2 and NMC-P3 may be extracellularly accessible on cancer cells for binding. In similar experiments, two HDM2-specific monoclonal antibodies that did not react with intact cancer cells were identified (FIG. 15B). One mAb was an anti-HDM2 antibody termed “Anti-MDM2 (Ab-4) Mouse mAb (2A9C1.18)” (EMD Millipore, Cat. No. OP144), which is raised against amino acid 153 to 222 of full length HDM2 (SEQ ID NO:4) that includes one Nuclear Localization Signal (NLS) and one Nuclear Export Signal (NES). Another HDM2-specific mAb that did not react with intact cancer cell membrane was an anti-HDM2 antibody termed “Anti-MDM2 (Ab-1) Mouse mAb (IF2)” (EMD Millipore, Cat. No. OP46), which is raised against amino acids 26-169 of HDM2 (FIG. 15B).

8.9 Example 9: In Vivo Anti-Tumor Effect of Antibodies that Target the Extracellularly Accessible Sequences of HDM2 in Cancer Cells

As demonstrated above, there are select epitopes of HDM2 that are extracellularly accessible on cancer cells. Described herein are three (3) such extracellularly accessible segments of HDM2, namely NMC-P1 (SEQ ID NO:1), NMC-P2 (SEQ ID NO: 2) and NMC-P3 (SEQ ID NO: 3). Data herein demonstrated that select antibodies raised against these 3 segments selectively and specifically bound to various types of cancers but not normal health cells (FIGS. 3-9). Moreover, results presented herein demonstrated the extracellular accessibility of these 3 segments of HDM2 on cancer cells (FIGS. 10 and 11). Furthermore, data herein show that HDM2-specific antibodies had both growth inhibitory and cytotoxic effect against cancer cells in vitro. Finally, the anti-tumor activity of HDM2-specific antibodies was evaluated in vivo. As an example, efficacy of mAb NMC-103 and NMC-204 on lung, colon and pancreatic cancer was tested.

Methodology used (Syngeneic Mouse Models of lung, colon and pancreatic cancer): To investigate the anti-tumor effects of HDM2-specific antibodies on lung, colon and pancreatic tumors in vivo, three well-known syngeneic mouse models of lung, colon and pancreatic cancer were used (Sharma et al., 1999, J. Immunol. 163 (9):5020-5028; McIntyre, 2015, Bioassays 37(8):909-920; Li, et al., 2015, Sci. Rep. 5:7856). Subcutaneous tumors were prepared by implanting either mouse Lewis Lung cancer LL/2 cells (2.5×10⁵ cells/mouse), mouse colon cancer MC-38 cells (5×10⁵ cells/mouse) or mouse pancreatic cancer Panc-2 cells (2×10⁶ cells/mouse) in the right flanks of 8-week-old female C57BL/6 mice (n=6-7). The mice were returned to their respective cages after tumor implantation. From day 7 after tumor implantation, the tumor volumes were measured 2 times/week with a digital caliper. In the case of MC-38 and Panc-2 study, treatment started when the tumors reached an average of 70-80 mm³. In the case of LL/2 study, tumor cell inoculation and treatment were initiated simultaneously. Mice were micro-chipped and registered following tumor implantation. Tumor volume measurements and body weights were recorded and mice were randomized into groups that received: A) mAb NMC-103 at 0.4 mg/kg (3 times a week for 3 weeks), 2 mg/kg (2 times a week for 3 weeks), or 4 mg/kg (2 times a week for 3 weeks); B) NMC-204 at 0.4 mg/kg (3 times a week for 3 weeks); C) isotype control mouse IgG1 (Abcam, Cat. No. ab18447) or IgG3 (Abcam, Cat. No. ab18392) at 0.4 mg/kg (3 times a week for 3 weeks) or 4 mg/kg (2 times a week for 3 weeks); D) Gemcitabine (25 mg/kg) and nab-paclitaxel (5 mg/kg) 2 times a week for 3 weeks; or E) a combination of gemcitabine (25 mg/kg), nab-paclitaxel (5 mg/kg) and mAb NMC-103 (2 mg/kg; 2 times a week for 3 weeks) or (10 mg/kg; 2 times a week for 2 weeks). All tumors were injected subcutaneously and all treatments were performed by intraperitoneal injection. Any mouse displaying prolonged adverse clinical signs, or with body weight loss exceeding 15% relative to body weight at day 0, was euthanized. Tumor measurements from post-treatment with HDM2-specific antibodies versus isotype control mouse antibodies versus standard of care treatment (gemcitabine and nab-paclitaxel) were made, and results are presented in FIGS. 16-19.

FIG. 16 demonstrates the anti-tumor efficacy of mAb NMC-204 (0.4 mg/kg 3 times a week for 3 weeks; dashed line), in mice that were simultaneously inoculated with LL/2 Lewis Lung cancer cells. As presented in the tumor volume measurement graph, by day 21, treatment with NMC-204 reduced tumor volume (1446 mm³) as compared with treatment with isotype control mouse IgG3 (2138 mm³). These results demonstrate the efficacy of an anti-HDM2-specific antibody (i.e., mAb NMC-204) that targets extracellularly accessible epitopes of HDM2 on lung cancer cells. For future patient treatment, the dosing and method of administration can be further expanded, e.g., to oral or intravenous delivery of the drug for optimum effect.

FIGS. 17 and 18 demonstrate the efficacy of mAb NMC-103 and NMC-204 against MC-38 syngeneic mouse model of colon cancer, respectively. As presented in FIG. 17A, mice treated for 18 days with mAb NMC-103 (0.4 mg/kg; 3 times a week for 2.5 weeks; dashed line) grew to 1268 mm³ while mice treated with isotype control mouse IgG1 reached a tumor volume of 2205 mm³ (solid line). Moreover, immunohistochemical staining for Ki67 protein, a well-established cell proliferation marker (Li et al., 2015, Mol. Med. Rep. 11(3):1566-72), revealed that mice treated with mAb NMC-103 had only 5% of tumor cells that stained positive for Ki67, while 80% of tumors in mice treated with isotype control antibody stained positive for Ki67 (FIG. 17B). These results further confirm and are consistent with the growth inhibition result observed in vitro (FIG. 12). Furthermore, the anti-tumor efficacy of another HDM2-specific antibody, mAb NMC-204, that recognizes a different extracellularly accessible segment of membrane HDM2, namely NMC-P2 (SEQ ID NO:2), was evaluated. As shown in FIG. 18A, tumor volume in mice treated with mAb NMC-204 (0.4 mg/kg; 3 times a week for 3 weeks) grew to 1670 mm³ while tumor in the control antibody-treated group reached 2555 mm³ in volume. Furthermore, FIG. 18B shows that mice treated with NMC-204 had 30% of their tumor cells stained positively for Ki67 while mice in the control group had approximately 80% of their tumor cells staining positively for Ki67, demonstrating the anti-proliferative effect of NMC-204 treatment on tumor growth.

The efficacy of HDM2-specific antibody treatment and standard of care chemotherapy was further compared. Utilizing the Panc-2 syngeneic mouse model of pancreatic cancer, FIG. 19 demonstrates the synergistic effect of NMC-103 in combination with pancreatic cancer standard-of-care treatment drugs: Gemcitabine (G)+nab-Paclitaxel (nP). As shown in the tumor volume graph of FIG. 19, when tumors in mice reached approximately 70 mm³, mice were divided into 4 groups (n=6) that received: A) isotype control mouse IgG1 (2 mg/kg); B) low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg); C) NMC-103 (2 mg/kg); or D) a combination of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103 (2 mg/kg). All drugs were injected intraperitoneally 2 times a week for 3 weeks. After receiving the 6^(th) dose on day 19, treatments were stopped for 2 weeks. By day 35, mice in groups that received either isotype control IgG (group A) or a combination of low dose Gemcitabine and nab-Paclitaxel (group B) reached an average of 2175 mm³ (open triangle) and 2314 mm³ (open circle), respectively. Both of these groups had reached morbidity criteria and were terminated. By day 35, tumors in mice of group C that received mAb NMC-103 (2 mg/kg) only reached on average 1523 mm³ (filled triangle), whereas tumors in mice of group D that had received a combination of NMC-103 (2 mg/kg), Gemcitabine and nab-Paclitaxel were measured at an average of 797 mm³ (filled circle). These two groups (groups C and D) of mice were then treated with a combination of NMC-103 (10 mg/kg), Gemcitabine and nab-Paclitaxel two times a week for 2 weeks. As demonstrated in FIG. 19, within 2 weeks tumors in both groups (C and D) reached the point where no measurable tumor was found.

The anti-tumor efficacy of NMC-103 and of its combination with pancreatic cancer standard of care treatment Gemcitabine (G) and nab-Paclitaxel (nP) was further evaluated in vivo utilizing the Panc-2 syngeneic mouse model of pancreatic cancer discussed above. In this Panc-2 study, treatment started after Panc-2 cell inoculation and when the tumors reached 80-100 mm³, with mAb NMC-103 administered at 10 mg/kg, two times a week in a larger number of mice (n=8/group) than that used for the study of FIG. 19.

FIG. 27 shows the results of a study wherein when Panc-02 tumors reached 80-100 mm³ in size, mice were randomly divided into 4 groups (n=8/group) that received: A) isotype control mouse IgG1 (10 mg/kg); B) low dose Gemcitabine (25 mg/kg) and nab-Paclitaxel (5 mg/kg); C) NMC-103 (10 mg/kg); or D) a combination of low dose Gemcitabine (25 mg/kg), nab-Paclitaxel (5 mg/kg) and NMC-103 (10 mg/kg). All drugs were injected intraperitoneally 2 times a week for 4 weeks. By day 30, mice in group A that had received isotype control antibody reached an average tumor size of 2028 mm³ and were terminated. By day 34, half of the mice in group B that had received low dose of the standard of care G & nP chemotherapy had died (n=4) and the tumor in the other half (n=4) had reached an average size of 1654 mm³. In contrast, all mice (n=8) in group C that had received NMC-103 were alive with average measurements of 230 mm³ that was due to scard and inflammation at the site of initial tumor inoculation. On the other hand, in group D that received NMC-103 in combination with G+nP, one mouse had died and the average measurements for the rest of the mice in that group (n=7) was 117 mm³ that was due to scars and inflammation at the siste of tumor inoculation. Both mice in groups C (n=8) and D (n=7) were then kept for another 4 weeks (62 days since the start of the study) with no further drug treatments during which time no sign of tumor growth beyond the initial scar size was observed, which demonstrated the lack of tumor in these mice.

As shown in FIG. 28, a Kaplan Meier survival analysis demonstrated significant survival benefit in mice that received NMC-103 alone or in combination with chemotherapy when compared to chemotherapy alone or control antibody under the experimental conditions described in FIG. 27. This study confirms the observations discussed above in connection with FIG. 19 and the ability of NMC-103 antibody to be used as single arm or in combination with chemotherapy.

To further evaluate the underlying immunological response of the long-term anti-tumor effect of NMC-103, at 62 days after the start of the study, mice that had previously received a combination of G+nP, mice in group C (mice that had previously been treated with NMC-103) and group D (mice that had been treated with a combination of NMC-103+G+nP), as described in FIG. 27, were re-challenged by a second round of Panc-2 inoculation (subcutaneous injection of 2×10⁶ cells/mouse), on the left dorsal flank. Tumor growth was monitored for 10 days at which point, a tumor of 90 mm³ (FIG. 29) was measured in the mice from group B. In contrast, as shown in FIG. 29, no tumor was observed in mice from the two groups that had previously received NMC-103 antibody (Groups C and D). These studies demonstrate the ability of antibodies that target the extracellularly accessible epitopes of M(H)DM2/4 to robustly activate the host immune system and provide long-term anti-tumor immunity against cancer.

To further establish the effectiveness of NMC-103 antibody in the treatment of large size tumors (i.e. advanced cancers), in another Panc-2 study (with results shown in FIG. 30), mice were treated with pancreatic cancer standard of care (Gemcitabine (25 mg/kg)+nab-Paclitaxel (5 mg/kg)) for 19 days at which point the tumor reached a size of approximately 450 mm³. The mice were then randomly divided in 2 groups that received a single dose of: A) isotype control mouse IgG1 (10 mg/kg) or B) NMC-103 (10 mg/kg). As shown in FIG. 30, a single i.p. injection of NMC-103 reduced the tumor size by almost half 6 days post treatment (from 438 mm³ to 233 mm³).

To further evaluate the NMC-103 dose-dependent anti-tumor effect, in a separate study, the MC-38 syngeneic tumor in mice (n=5) was re-established and treatment carried out similarly to the study of FIG. 17 with the following modification: increase of antibody concentration from 0.4 mg/kg to 10 mg/kg. As shown in FIG. 31, mice treated with NMC-103 at 10 mg/kg, two times per week for three weeks resulted in a greater tumor reduction when compared to mice treated with NMC-103 at 0.4 mg/kg (FIG. 17). These data support the dose-dependent anti-tumor effect of antibodies that bind to extracellularly accessible epitopes of M(H)DM2/4.

Utilizing another syngeneic mouse model of colon cancer, CT-26 (Selby et al., 2016, PLoS One. 9; 11(9): e0161779), the anti-tumor efficacy of a chimeric version of monoclonal antibody NMC-303 was assessed. To create the chimeric version of monoclonal antibody NMC-303, isotype class-switching was performed on a mouse NMC-303 (having heavy variable regions of SEQ ID NO:40 and light chain variable regions of SEQ ID NO:41) to convert it from a mouse IgM to a chimeric IgG1. The mouse Heavy and Light chain variable regions (SEQ ID NO:40 and SEQ ID NO:41, respectively) were cloned into a human Ig gamma-1 chain and human Ig kappa chain as constant region. A total of eight (8) BALB/c mice were injected subcutaneously with CT-26 (800,000 cells/mouse). Mice were then divided into two groups (n=4) that received: A) control antibody (10 mg/kg) or B) the chimeric version of monoclonal antibody NMC-303 (10 mg/kg) two times a week for 3 weeks. FIG. 32A shows that by day 24 post tumor inoculation, mice treated with the chimeric version of monoclonal antibody NMC-303 (10 mg/kg) reached an average tumor size of 726 mm³, while mice treated with control antibody (10 mg/kg) had an average tumor size of 1746 mm³. Furthermore, FIG. 32B shows the individual mouse tumor sizes on day 24 post tumor inoculation. Due to the human constant region of the chimeric version of the monoclonal antibody NMC-303, the anti-tumor efficacy of this antibody might be improved if tested in mouse models with human immune background. The above in vivo results further support the anti-tumor efficacy of antibodies raised against NMC-P3 immunogen that target the extracellularly accessible epitopes of the M(H)DM2/4 on cancer cells.

Taken together, these results demonstrate two important therapeutic aspects of M(H)DM2/4-specific antibodies: First, anti-M(H)DM2/4 antibodies that target extracellularly accessible sequences of M(H)DM2/4 are in themselves effective anti-cancer agents. Second, M(H)DM2/4 antibodies used in combination with low concentrations of chemotherapeutics have a potently synergistic anti-tumor effect. This is particularly important, considering the frequently observed side-effects and limitations of chemotherapy used at their clinically effective concentrations. In addition, these results demonstrate that M(H)DM2/4 antibodies lead to development of long-term anti-tumor immunity against cancer as demonstrated by prevention of recurrence of cancer and long-term survival of animals previously treated with M(H)DM2/4 antibodies.

The in vitro data showed an increase in plasma membrane M(H)DM2/4 when cells are treated with various types of chemotherapeutic agents such as Gemcitabine and Paclitaxel (data not shown). This treatment potentially makes those cells more susceptible to anti-HDM2 antibody therapy, which further explains the synergistic effect of their combinatorial administration (FIG. 19).

8.10 Conclusions Based on Examples 1-9

Taken together, the data presented herein demonstrate that select M(H)DM2/4-specific antibodies that recognize extracellularly accessible segments of M(H)DM2/4 in cancers cell have growth inhibitory and cytotoxic effect against a variety of cancers while sparing normal untransformed cells. The selective anti-tumor effect of these antibodies is believed to be due at least in part to their recognition of extracellularly accessible epitopes of M(H)DM2/4 protein variants that are expressed on the surface of cancer cells while the expression levels of these M(H)DM2/4 variants on the cell surface of normal cells are low or absent.

Examples 10-12 below describe data obtained using other anti-HDM2 antibodies that bind to segments of HDM2 that are extracellularly accessible on cancer cells.

8.11 Example 10: Other HDM2-Specific Antibodies that Also Bind to Intact Cells from Different Human Cancer Cell Lines and Freshly Isolated Primary Human Cancer Cells but not to Normal Cells

To further extend the therapeutic and diagnostic potentials of other antibodies raised against M(H)DM2/4 we evaluated several other available antibodies for their binding and anti-tumor activity. As described above, some of these antibodies (i.e. Calbiochem/Millipore OP-46 and OP-144) did not react with extracellularly accessible epitopes of M(H)DM2/4, demonstrating lack of epitope availability on cancer cell membranes, indicating that not all anti-M(H)DM2/4 antibodies can be used for the treatment of cancer. However, several other anti-M(H)DM2/4 antibodies were shown to not only interact with extracellularly accessible epitopes on the cancer cell membrane, but also to have in vitro and/or in vivo anti-tumor activity.

The following anti-HDM2 antibodies were used in the experiments described in Examples 10-12: (i) polyclonal sc-813, N-20, rabbit IgG, from Santa Cruz (abbreviated throughout the specification as “N-20” or “sc-813 (N-20)”); (ii) monoclonal OP145, mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP145”); (iii) monoclonal OP46 (Ab-1), mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP46”); (iv) monoclonal OP144 (Ab-4), mouse IgG1, from Calbiochem (abbreviated throughout the specification as “OP144”); (v) polyclonal sc-812, C-18, rabbit IgG, from Santa Cruz (abbreviated throughout the specification as “C-18” or “sc-812 (C-18)”); and (vi) monoclonal 965 (SMP14), mouse IgG1, from Santa Cruz (abbreviated throughout the specification as “SMP14” or “965 (SMP14)”). Table 10, below, provides information regarding the HDM2 recognition sites of these antibodies (i.e., amino acids of HDM2 recognized by these antibodies), and whether or not these antibodies are cytotoxic to cancer cells. OP145, N-20, C-18 and SMP14 were cytotoxic to cancer cells tested, and OP46 and OP144 were not cytotoxic to cancer cells tested.

Utilizing Fluorescence-activated cell sorting (FACS), the data presented herein show that select HDM2-specific antibodies bind to the surface membrane of live human cancer cells, human melanoma A2058 cells maintained in culture, and two primary patient-derived ovarian cancer cells OVCA-1 and OVCA-4 that had been freshly isolated from ovarian cancer tissues. In contrast, FACS analysis of live normal mouse spleenocytes demonstrated the absence of plasma membrane staining with the same HDM2-specific antibodies.

Methodology used: Intact cells released either with either EDTA or Trypsin were blocked with 5% human serum albumin. Cells were then incubated with the either polyclonal (N20) or monoclonal (OP145) HDM2-specific antibodies for 90 min on ice. Another set of cells prepared under the same conditions were incubated with the same antibodies that were pre-incubated with their corresponding blocking peptides before incubation with cells. Following primary antibody incubation, cells were washed 3 times with ice-cold PBS followed by FITC-secondary antibody incubation for 60 min. Cells were then washed 3 times with PBS and were subjected to FACS analyzer.

FIG. 24 presents results of the FACS analysis of human melanoma, primary ovarian cancer, and normal mouse spleenocytes. FIG. 24A: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. FIG. 24B: area under curve #1 represents cells incubated with goat anti-mouse secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 monoclonal antibody OP145 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 monoclonal antibody OP145 followed by goat anti-mouse secondary antibody. FIG. 24C: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N20 followed by goat anti-rabbit secondary antibody. FIGS. 24D & E: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #3 represents cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody. FIG. 24F: area under curve #1 represents cells incubated with goat anti-rabbit secondary antibody only; area under curve #2 represents trypsin-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody; area under curve #3 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 pre-incubated with its blocking peptide followed by goat anti-rabbit secondary antibody; area under curve #4 represents EDTA-released cells incubated with anti-HDM2 polyclonal antibody N-20 followed by goat anti-rabbit secondary antibody.

EDTA-released intact human melanoma A2058 cells incubated with either an anti-HDM2 polyclonal N-20 antibody (FIG. 24A; area under the curve #3) or monoclonal OP145 antibody (FIG. 24B; area under the curve #3) show cells that are stained positive for HDM2 on their cell surface. To control for epitope-specificity, no specific staining beyond background was observed when either one of these antibodies was pre-incubated with its corresponding blocking peptide prior to incubation with the cells (see FIGS. 24A and 24B; area under the curve #2). Interestingly, in the case of human melanoma cells released using trypsin, which cuts the external portion of cell surface proteins, no cell surface staining of HDM2 was observed (see FIG. 24C; area under the curve #3 when compared with cells released with EDTA, area under the curve #4), further indicating the presence of at least parts of the HDM2 protein on the external face of the plasma membrane. Moreover, freshly isolated tumors from two patients with ovarian cancer (OVCA-1 and OVCA-4) showed extensive surface staining when incubated with the polyclonal N-20 antibody (see FIGS. 24D and 24E; area under the curve #3). This staining was completely blocked when the antibody was pre-incubated with its blocking peptide prior to incubation with the cells (see FIGS. 24D and 24E; area under the curve #2). However, intact mouse normal spleenocytes did not stain over the background with the same antibodies (see FIG. 24F; compare staining when incubated with HDM2-specific N-20 antibody in area under the curve #4, with staining in samples treated with the antibody pre-incubated with N-20 blocking peptide in area under the curve #3). Taken together, the results of FACS analysis strongly indicate the presence of extracellularly accessible epitopes of M(H)DM2 on the plasma membrane of the intact cancer cells and its absence in normal untransformed cells.

8.12 Example 11: Certain HDM2-Specific Antibodies Initiate Complement-Mediated Cytotoxicity Against Different Types of Cancers

The data presented herein show the ability of select HDM2-specific antibodies to not only bind to the surface membrane of cancer cells but also to initiate a cytotoxic effect in the presence of fresh normal human serum (NHS). The cytotoxic effect was measured in various cancer cells such as human melanoma, pancreatic, breast and ovarian cancer cells as well as in normal human fibroblasts and blood cells.

Methodology used: Cells seeded in 24-well dishes were grown over night at 37° C. in a humidified atmosphere supplied with 5% CO₂-95% air mixture. The following day the cells were thoroughly washed with ice-cold PBS and incubated with various HDM2-specific antibodies (i.e., OP145, N-20 and C-18 antibodies; see description of the antibodies in Table 10) or control antibodies (directed to the intra-cellular protein Cytochrome C) diluted in culture medium containing 1% BSA to a final antibody concentration of 5-10 ug/ml. After 30 min, NHS was added to the cells to make a final concentration of at 1:30 in the culture medium with 1% BSA together with 1% propidium iodide (PI) solution. As controls for the role of complement in the fresh human serum, parallel cultures treated with HDM2-specific antibodies were exposed to fresh human serum that had been incubated for 30 min at 56° C. (HiNHS), a process that is known to disenable complement activity. Images were taken at 15, 30, 45 and 60 min post incubation using Olympus FluoView FV1000 Confocal Laser Scanning Biological Microscope built on the Olympus IX81 Inverted Microscope.

FIG. 25 demonstrates the cytotoxic effect of HDM2-specific antibodies against human pancreatic and ovarian cancer cells (FIG. 25A), and rodent pancreatic cells (FIG. 25B). OP145, N-20 and C-18 antibodies in the presence of NHS trigger complement-mediated cytotoxicity in cancer cells incubated with such antibodies, resulting in the death of the cancer cells as evident by the nuclear uptake of the cell-death marker Propidium Iodide (PI) (see FIG. 25A, panels b and e, for ovarian cancer cells incubated with OP145 and FIG. 25B, panels b and c, for pancreatic cancer cells incubated with N-20 and C-18, respectively). In the presence of control antibodies (i.e., anti-cytochrome C antibody, see FIG. 25A, panel c, and FIG. 25B, panel e) cancer cell death is similar to that of control NHS alone, without any antibodies (see FIG. 25A, panels a and d, and FIG. 25B, panel a) or normal cells (Fibroblasts, FIG. 25A, panel g) treated with HDM2-specific antibodies (i.e., there is no or minimal cell death as indicated by lack of PI staining, se FIGS. 25A, panels a, c, d, f and g, and FIG. 25B, panel 1). Further, no cytotoxicity was observed when cells were treated with anti-HDM2 monoclonal OP46 antibody (see FIG. 25B, panel d). FIG. 25C provides quantitative representations of HDM2-specific antibody-dependent complement cytotoxicity against human pancreatic cancer cells. Cells treated with anti-HDM2 (C-18) antibody in the presence of NHS demonstrated cytotoxicity over 15-30 min. post-treatment, whereas anti-HDM2 OP46 shows no cytotoxic effect beyond that observed when cells were treated with control anti-Cytochrome C antibody or when cells were left untreated in the presence of NHS.

Moreover, HDM2-specific antibodies in the presence of heat-inactivated human serum (i.e., fresh human serum that had been incubated for 30 min at 56° C. (HiNHS)) did not have any cytotoxic effect on cancer cells (i.e., did not result in the death of the cancer cells as was evident by PI staining), demonstrating that the cytotoxic effect is due to complement activity.

Table 10 lists antibodies tested by the inventors and summarizes results obtained relating to the in vitro cytotoxic effects of various anti-HDM2 antibodies against cancer cells.

TABLE 10 Antibodies used and their ability to induce a cytotoxic effect in cancer cells in vitro. HDM2 Recognition Site Company Amino Acid Nos. Species Antibody Purchased M(H)DM2 Abs of SEQ ID NO: 4 Cytotoxicity Reactivity* Host Type From OP46 (Ab-1)  26-169 − H Mouse IgG1 Monoclonal Calbiochem OP144 (Ab-4) 153-222 − H & M Mouse IgG1 Monoclonal Calbiochem OP145 19-50 + H & M Mouse IgG1 Monoclonal Calbiochem sc-813 (N-20) N-terminus + H & M Rabbit IgG Polyclonal Santa Cruz sc-812 (C-18) C-terminus + H, M, R Rabbit IgG Polyclonal Santa Cruz 965 (SMP14) 154-167 + H, M, R Mouse IgG1 Monoclonal Santa Cruz Control Antibodies Cytochrome C N/A − H Mouse IgG1 Monoclonal Santa Cruz Histone H4 N/A − Broad Mouse Monoclonal Santa Cruz Species IgG_(2a) Beta-Tubulin N/A − H, M, R Rabbit IgG Polyclonal Santa Cruz *H—binds to human protein; M—binds to murine protein; R—binds to rabbit protein

8.13 Example 12: Results Regarding In Vivo Anti-Tumor Effect of Other Antibodies that Bind to Extracellularly Accessible Epitopes of HDM2

To further evaluate the anti-cancer activity of mAbs raised against extracellularly accessible epitopes of HDM2 (other than NMC-P1, NMC-P2 and NMC-P3) on cancer cells, studies of the anti-tumor efficacy of anti-HDM2 monoclonal antibody OP-145 (Calbiochem) and “MDM2 monoclonal antibody (M01), clone 1A7” (Abnova, Cat. No. H00004193-M01) were conducted. As demonstrated above, neither one of these antibodies showed reactivity to NMC-P1 and NMC-P2 (see FIGS. 14A-B), but both of these antibodies showed reactivity with intact cancer cells (see FIGS. 15A and 24B).

Evaluation of anti-tumor effects of antibody OP145 on pancreatic tumor growth in vivo: Subcutaneous tumors were prepared by implanting Panc02 cells (2×106 per mouse) in the right flanks of female 8-week-old C57BL/6 mice. The mice were returned to their respective cages after tumor implantation. From day 7 after tumor implantation, the tumor volumes were measured 2 times/week with a digital caliper. Treatment started when the tumors reached an average of 200 mm³ (see FIG. 26, day 14). Mice were micro-chipped and registered following tumor implantation. Tumor volume measurements and body weights were recorded and mice were randomized into two groups that received either OP145 antibody (by intra-tumoral injection of 0.1 mg/kg of mouse monoclonal antibody OP145, 3 times per week) or PBS. Three mice were treated with OP145, and five mice were treated with PBS. Any mouse displaying prolonged adverse clinical signs, or with body weight loss exceeding 15% from Day 0 body weight, was euthanized. Tumor measurements from the first 10 days post treatment with OP145 antibody versus PBS were made, and results are represented in FIG. 26.

It was found that subcutaneous tumors in mice administered the OP145 antibody had a size of 260 mm³ at 21 days post tumor cell injection, whereas subcutaneous tumors in mice administered PBS had a size of 375 mm³ (see FIG. 26).

In vivo studies of the effect of “MDM2 monoclonal antibody (MOO, clone 1A7” against MC-38 syngeneic mouse model of colon cancer (using 0.4 mg/kg of the M01, clone 1A7 antibody; 3 times a week for 3 weeks, intraperitoneally; n=4) demonstrated that while the antibody-treated mice initially responded to the treatment (average tumor size after 6 doses was at 1588 mm³ in antibody-treated group vs 1999 mm³ in isotype control-treated group), tumors in both control and antibody-treated mice reached approximately 2500 mm³ by third week (FIG. 23). During the same time period, mAb NMC-103 and NMC-204 given to mice at the same dose (0.4 mg/kg; 3 times a week for 3 weeks, intraperitoneally) demonstrated a significant (p<0.005) reduction in tumor volume with lasting anti-tumor activity (FIG. 19; open circle and open square, respectively; also see FIGS. 17A and 18A). This finding indicates that although mAb M01 had strong binding to cells in vitro, the anti-tumor effect of NMC-103 and NMC-204 was superior to that of mAb M01 (FIG. 23).

It must be noted that these results relating to in vivo anti-tumor efficacy of monoclonal antibodies OP-145 and M01 must be interpreted with caution since the commercial antibody preparations used may not necessarily have the desired clonality, purity or pharmaceutically acceptable formulation for in vivo efficacy testing.

9. EXAMPLES 13-18

The data presented in Examples 13-18 herein shows the binding of humanized and chimeric antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3 of plasma membrane-bound M(H)DM2 to intact, non-permeabilized cancer cells, as demonstrated by the binding epitope availability of plasma membrane markers (i.e. E-Cadherin) and the absence of binding of antibodies to intracellular markers (Cytochrome-C, Cyclin D1 and Bcl2).

In addition, data presented herein shows reactivity of humanized and chimeric antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3 of plasma membrane-bound M(H)DM2 in several human and rodent cancer cell lines as well as primary human tumors.

Furthermore, the binding of humanized and chimeric antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3 of plasma membrane-bound M(H)DM2 is reduced when the binding of the antibodies is competed with their respective immunogenic peptides. Moreover, antibody-antigen binding is abolished when surface proteins are enzymatically digested in otherwise intact cells, demonstrating the extracellular availability of the binding epitopes.

The in vivo anti-tumor activity of the humanized and chimeric antibodies was also tested. Data presented here demonstrates the in vivo anti-tumor activity of humanized and chimeric antibodies to plasma membrane-bound M(H)DM2 and show that the anti-cancer efficacy is dependent on stimulation of the immune system.

Finally, in vitro and in vivo data presented here demonstrate, for the first time, the correlation between MDM2 gene amplification observed in hyper-progressive patients under immunotherapy and increased expression of plasma membrane-bound M(H)DM2 on the surface of the hyper-progressive tumors. This observation further demonstrates the rationale for the therapeutic utilization of antibodies that target extracellularly accessible epitopes of plasma membrane-bound M(H)DM2 for treatment of patients who exhibit hyper-progression of cancer in response to administration of an immunotherapy.

Antibodies Used in Examples 9-13

The following anti-HDM2 antibodies were used in the experiments described in Examples 13-18: (i) chimeric NMC-103 (“NMC-C103.VH0/VK0” or “NMC-C103”) monoclonal antibodies, (ii) humanized variants of NMC-C103 (“Humanized NMC-103” or “NMC-H103”), and (iii) chimeric NMC-303 (“NMC-C303”) monoclonal antibodies as described below. Chimeric NMC-103 (“NMC-C103.VH0/VK0” or “NMC-C103”) monoclonal antibodies were generated by replacing the constant region of mouse IgG1 monoclonal antibody NMC-103 with a human IgG1 constant region. As described above, a monoclonal antibody NMC-103 is an antibody that binds to NMC-P1 (SEQ ID NO:1) (it is one of the NMC-100s series of antibodies). Humanized variants of NMC-C103 (“Humanized NMC-103” or “NMC-H103”) were then designed comprising combinations of the following variable heavy and light chains: VH0, VH1, VH2, VH3, VH4, VH6, VH7 and VK0, VK1, VK2, VK3, VK4, VK5, VK6, VK7, correspondingly (see Tables 13-27 and SEQ ID NOs: 283, 287, 291, 295, 299, 305, 309 and 285, 289, 293, 297, 301, 303, 307, 311, respectively). Immunoglobulin class switching was used to generate chimeric NMC-303 (“NMC-C303”) monoclonal antibodies, where the constant region of NMC-303 mouse IgM monoclonal antibody underwent isotype-switching with human IgG1 constant region (Tables 11 and 12 and SEQ ID Nos: 40 and 41). As discussed above, a monoclonal antibody NMC-303 is an antibody that binds to NMC-P3 (SEQ ID NO:3) (it is one of the NMC-300s series of antibodies). The heavy chain/light chain framework region sequences, complementarity determining region (CDR) sequences, and variable region sequences of these antibodies are listed in Section 11, below.

Methods of Making of Antibodies Used in Examples 9-13

Generation of humanized and chimeric antibodies: Design of Composite Human Antibody™ Variable Region Structural models of the murine antibody variable (V) regions were produced using Swiss PDB and analyzed in order to identify important “constraining” amino acids in the V regions that were likely to be essential for the binding properties of the antibody. Most residues contained within the CDRs (using both Kabat and Chothia definitions) together with a number of framework residues were considered to be important. The VH and VK sequences of murine mAb contain typical framework residues and the CDR 1, 2 and 3 motifs are comparable to many murine antibodies. From the above analysis, it was considered that Composite Human sequences of mAbs could be created with a wide latitude for alternative residues outside of the CDRs but with only a narrow menu of possible residues within the CDR sequences. Preliminary analysis indicated that corresponding sequence segments from several human antibodies could be combined to create CDRs similar or identical to those in the murine sequences. For regions outside of, and flanking the CDRs, a wide selection of human sequence segments were identified as possible components of the novel humanized V regions.

CD4+ T Cell Epitope Avoidance: Based upon the structural analysis, a large preliminary set of sequence segments were identified that could be used to create humanized variants. These segments were selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MEW class II alleles (Perry et al., 2008) and using the TCED™ of known antibody sequence-related T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II or that scored significant hits against the TCED™ were discarded. This resulted in a reduced set of segments, and combinations of these were again analyzed, as above, to ensure that the junctions between segments did not contain potential T cell epitopes. Selected sequence segments were assembled into complete V region sequences that were reduced in significant T cell epitopes. Six heavy chain (VH1 to VH4, VH6, and VH7) and seven light chain (VK1 to VK7) sequences were chosen for gene expression in mammalian cells.

Construction, Transfection and expression of Chimeric Antibody and Humanized variants: The VH and VK sequences of murine mAb together with the humanized variants were synthesized with flanking restriction enzyme sites for cloning into expression vector system for human IgG1 heavy and kappa light chains. All constructs were confirmed by sequencing. DNA constructs encoding chimeric antibodies and humanized antibody variants having combinations of various heavy and light chain sequences were initially transiently transfected into CHO cells using a MaxCyte STX. Electroporation system (MaxCyte Inc., Gaithersburg, USA). Transfections were undertaken for each antibody using OC-400 processing assemblies. Following cell recovery, cells were pooled and diluted to 3×106 cells/mL in CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM LGlutamine (ThermoFisher, Loughborough, UK) and 1× Hypoxanthine-Thymidine (ThermoFisher, Loughborough, UK). Twenty four hours post transfection, the culture temperature was reduced to 32 ÅãC and 1 mM sodium butyrate (Sigma, Dorset, UK), 30% volume of CD-Efficient Feed B (ThermoFisher, Loughborough, UK), and 3.3% Volume of FunctionMax Titre Enhancer was added. An additional 15% (of the total volume) of CD Efficient Feed B and 1.65% FunctionMax titre enhancer was added at day 7 post transfection. Culture supernatants were harvested around 10-12 days post transfection.

Purification of humanized and chimeric antibodies: Humanized and chimeric variant antibodies were purified from cell culture supernatant using a 1 mL Mab Select Sure column (GE Healthcare, Little Chalfont, UK). The column was washed using 1×DPBS and protein eluted using 0.1 M sodium citrate pH 3.0. Collected fractions were then pooled, buffer exchanged into PBS pH 7.4 and filter sterilized. Purified antibodies were then quantified by OD280 nm using an extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence and analyzed by reducing SDS-PAGE.

Antibodies and Reagents Used in Cell-Based ELISA, Flow Cytometry and In-Vivo Studies

The following commercial primary antibodies were used in the cell-based ELISA experiments: Cytochrome C (Santa-Cruz 13156), α-Bcl2 N19 (SC-492), α-cyclin D1 (SC-717), E-cadherin G10 (SC-8426). Secondary anti-mouse (A24524, Thermo-fisher) and Secondary anti-human (62-8420, Thermo-fisher) with HRP conjugate were used as secondary antibodies. TMB substrate (34028, Thermo scientific) and Stop solution (cat. No SS04, Life Technologies) was used in ELISA assay for development of chemoluminescence.

For flow cytometry, an anti-mouse fluorescently labeled Secondary antibody (Biolegend 405307), Anti-human fluorescently labeled Secondary antibody (Life technologies H10104) and 7-AAD (420404, Biolegend) were used.

For in-vivo studies, the following antibodies were used: anti-mouse PD-1 antibody (BioXCell, Cat. No. BE0146) and mouse isotype control antibody (BioXCell BP0083) and human isotype control (BioXCell, Cat. No. BP0297). Human PBMCs were purchased from Stem Cell Technologies (Cat. No. 70025).

Cell-Based ELISA

In a 96-well microplate, 15,000 cells/well were cultured overnight. The next day, unbound cells were washed off with sterile 1×PBS. The cells in each well were fixed with freshly prepared 4% buffered paraformaldehyde (pH 7.2) for 1 hour, followed by 3 washes with 1×PBS. The wells were then blocked with 5% BSA in PBS (100 μl/well) for 2 hours at room temperature. Microplate wells were then washed 5 times with 300 μl of ice cold 1×PBS. Monoclonal antibodies NMC-H103 (VH4/VK3) or NMC-C303 at 5 μg/mL in 1% BSA/PBS were then incubated with various cancer cells for 2 hours at room temperature. Wells were then washed with 300 μl of ice-cold 1×PBS for 5 times and corresponding secondary antibody HRP-GaH F(ab′)2 diluted 1:2000 or 1:4000 in PBS with 1% BSA were added at 100 μl/well for 1 hour at room temperature followed by washing 5 times with 300 μl of ice-cold 1×PBS. TMB Substrate Solution (1-Step™ Ultra TMB-ELISA, ThermoFisher, Cat. No. 34028) was then added at 50 μL to each microplate well and incubated at room temperature for 30 minutes. The reaction was stopped by addition of 50 μL of stop solution (ThermoFisher, Cat. No. SS04) to each well and absorbance of each well was measured at OD450 nm. The absorbance value of each experimental well was corrected for the absorbance value obtained from wells treated with isotype-identical mAbs included in each experiment.

Flow Cytometry

Cells that had been allowed to grow to about 80% confluency in 75 cm² tissue culture flasks were released with either EDTA (10 mmols; pH 7.2; 5 min, 37° C.) or Trypsin (Gibco TrypLE Express), washed several times with ice-cold PBS, and blocked for 20 minutes on ice with 5% human serum albumin in PBS (intact, non-permeabilized cells). After establishing viability, the cell number was adjusted to 10⁶ cells/ml and kept in ice-cold PBS until staining. Cells were then incubated with primary antibodies NMC-H103 (VH4/VK3) or NMC-C303 (1 μg/ml) for 30 minutes on ice. Following primary antibody incubation, cells were washed 3 times with ice-cold PBS, followed by incubation with goat anti-human PE labeled secondary antibody (Life technologies H10104) on ice for 20 minutes. Cells were then washed 3 times with PBS and incubated with 7AAD (5 μl/500 mL of buffer) before flow cytometry analysis using Fortessa 5.

MTT Cell Proliferation Assay

Cells were seeded in a 96-well plate at a density of 1000 cells/well in Serum free Media (Sigma Lifesciences Cat. No. S9388) and were simultaneously treated with anti-mouse PD-1 (BioXcell Cat. No. CD279) at the concentration of 75 μg/ml and 100 μg/ml and IgG control (Abcam Cat. No. ab18443) at 100 μg/ml. Cells were grown at 37° C. for 48 hours in a humidified CO2 incubator. On the 2^(nd) day of incubation, 15 μl of MTT Dye Solution (Promega Cat. No. G402A) was added to each well. The plate was incubated at 37° C. for 3 hours in a humidified CO2 incubator. 100 μl of Solubilization/Stop Solution (Promega Cat. No. G401A) was then added to each well. Plates were then read at absorbance at 570 nm using a 96-well plate reader (BioTek).

In Vivo Studies

Subcutaneous tumors were prepared by implanting either mouse colon MC-38 cells (5×10⁵ cells/mouse), human lung cancer A549 cells (5×10⁶ cells/mouse) or mouse lung cancer M109 cells (0.5×10⁵ cells/mouse) in the right flanks of NOD scid gamma mice (NSG: NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ) purchased from Jacksons Laboratory. From day 7 after tumor implantation, the tumor volumes were measured 2 times/week with a digital caliper. In the case of mouse colon MC-38 cells and human lung cancer A549 cells study in NSG mice, once tumors reached an average volume of 300-400 mm³, mice were randomly divided into 3 groups (n=4) and 7×10⁶ human PBMCs (Stem Cell Technologies Cat #70025) was injected into the tail vein of mice in groups 1 and 3. Seven days later, in the case of mouse colon MC-38 cells study, mAb NMC-C303 antibody (10 mg/kg) was injected intraperitoneally (i.p.) to mice in groups 2 and 3 and human isotype control antibody (BioXCell BP02) into mice in group 1 on days 33 and 35 post tumor inoculation. In the case of human lung cancer A549 cells study, seven days post PBMC injection, mice in group 2 and 3 received 1 intraperitoneal injection of mAb NMC-H103 (VH4/VK3) (10 mg/kg) while mice in group 1 received 1 injection of human isotype control antibody (BioXCell BP02) and tumors were measure 5 days later. In the case of mouse lung cancer M109 cells study, post subcutaneous injection of M109 cells, NSG mice were randomly divided into 2 groups. Group 1 mice received mouse isotype control antibody (12.5 mg/kg; once a week), while mice in group 2 received mouse anti-PD1 antibody (12.5 mg/kg; once a week) intraperitoneally or 5 weeks at which point tumors from mice in both groups were harvested for flow cytometry analysis.

Preparation of Single-Cell Suspension of Tumors

Preparation of single-cell suspension of the tumors harvested from mice was performed using MACS Tumor Dissociation Kit Mouse (Cat. no. 130-096-730) and manufacturer's protocol. Briefly, enzyme mix was prepared by adding 2.35 mL of RPMI, 100 μL of Enzyme D, 50 μL of Enzyme R, and 12.5 μL of Enzyme A into a gentle-MACS C Tube. Tumors were cut into pieces of 2-4 mm and transferred to gentle-MACS C Tune containing the enzyme mix and attached to gentle-MACS Dissociator. A pre-fix program was ran and samples were incubated for 40 minutes at 37° C. with continuous rotation using the MACSmix Tube Rotator. Tubes were re-suspended and the cell suspension was transferred to a MACS SmartStrainer (70 μm) placed on a 15 mL tube. Samples were washed with 10 mL of RPMI 1640 followed by centrifugation at 300×g for 7 minutes. Supernatant was then completely removed and the resulting single cells were re-suspended into Flow Cytometry staining buffer to be stained with desired antibodies.

9.1 Example 13: Humanized and Chimeric Antibodies to Extracellularly Accessible Epitopes NMC-P1 and NMC-P3 of the Membrane-Bound M(H)DM2 Bind to the Surface of Intact Cancer Cells

In Example 13, data of cell-based enzyme-linked immunosorbent assay (ELISA) experiments are presented showing that humanized mAb NMC-103 (NMC-H103; VH4/VK3) and chimeric mAb NMC-303 (NMC-C303) (both at 5 μg/mL) bind to intact mouse LL/2 cancer cells (average OD reading of 3.51 nm and 3.59 nm, respectively) (FIG. 33).

Results of the cell-based ELISA experiments showed that antibodies against intra-cellular proteins such as cytochrome-C, cyclin D1 and Bcl-2 (all at 5 μg/mL) showed no binding to intact mouse LL/2 cancer cells beyond background staining with secondary antibody only (OD<0.5 nm) (FIG. 33), while an antibody against the plasma membrane protein E-Cadherin (5 μg/mL) bound to its target (average OD reading of 1.49 nm) (FIG. 33), demonstrating intra-cellular target inaccessibility to antibodies in intact cells. The large size and ionic nature of antibodies preclude them from gaining intra-cellular access without membrane permeabilization (Muller S. et al, 2005). Considering the fact that antibodies are unable to cross the cell membrane of intact, un-permeabilized cells, antibodies to intra-cellular proteins are unable to bind their intra-cellular targets, while antibodies to surface proteins can bind their targets.

As shown in FIG. 33, similarly to the results shown in connection with plasma membrane protein E-Cadherin, mAb NMC-H103 (VH4/VK3) and mAb NMC-C303 (NMC-C303) bound to intact mouse LL/2 cancer cells, demonstrating that both of their target epitopes (i.e. NMC-P1 and NMC-P3) are extracellularly accessible on intact mouse LL/2 cancer cells.

9.2 Example 14: Humanized and Chimeric Antibodies to Extracellularly Accessible Epitopes NMC-P1 and NMC-P3 of the Plasma Membrane-Bound M(H)DM2 Bind to Various Intact, Un-Permeabilized Human and Murine Cancer Cells

Example 14 presents data of cell-based ELISA demonstrating that humanized mAb NMC-H103 (VH4/VK3) and chimeric mAb NMC-C303, which were raised against extracellularly accessible NMC-P1 and NMC-P3 epitopes of plasma membrane-bound M(H)DM2, respectively, bind to intact, un-permeabilized human (FIG. 34A) and murine (FIG. 34B) cancer cells.

As shown in FIG. 34A, cell-based ELISA experiments demonstrated the binding of mAb NMC-H103 (VH4/VK3) (5 μg/mL) and mAb NMC-C303 (5 μg/mL) to following human cancer cells: A2085 melanoma cells, MCF-7 breast cancer cells, MIAPaCa-2 pancreatic cancer cells, HeLa cervical cancer cells, OVCA-4 ovarian cancer cells, and OVCAR-3 ovarian cancer cells. In addition, as shown in FIG. 34B, cell-based ELISA experiments demonstrated the binding of mAb NMC-H103 (VH4/VK3) (5 μg/mL) and mAb NMC-C303 (5 μg/mL) to the following mouse cancer cells: MC-38 colon cancer cells and Panc-2 pancreatic cancer cells. No binding beyond background staining was shown using anti-human secondary antibody.

These data show the ability of mAb NMC-H103 (VH4/VK3) and NMC-C303 to react with extracellularly accessible epitopes of the plasma membrane-bound M(H)DM2 on intact human and rodent cancer cells.

9.3 Example 15: Humanized and Chimeric Antibody's Binding to Extracellularly Accessible Epitopes NMC-P1 and NMC-P3 of the Plasma Membrane-Bound M(H)DM2 on Human and Murine Cancer Cells is Specific and can be Competed with Epitope-Specific Peptides and Reduced by Enzymatic Digestion of Surface Target Antigen

To evaluate the specificity of humanized mAb NMC-H103 (VH4/VK3) to plasma membrane-bound M(H)DM2, a competition experiment with the mAb's immunogenic peptide was performed. FIG. 35A shows data from cell-based ELISA experiments demonstrating the binding of NMC-H103 (VH4/VK3) to intact, un-permeabilized mouse Lewis Lung LL/2 cancer cells with an OD reading of 3.48 nm, while pre-incubation of NMC-H103 (VH4/VK3) (5 μg/mL) with its specific immunogenic peptide (NMC-P1 at ×10 the concentration of mAbs), reduced the binding efficiency of mAb NMC-H103 (VH4/VK3) to its target (OD=1.09 nm). In contrast, pre-incubation of NMC-C303 with NMC-P1 had no effect on its binding to LL/2 cells, as mAb NMC-C303 has a different immunogenic peptide sequence (i.e. NMC-P3).

To distinguish live intact cells from permeabilized or dead cells, 7-Aminoactinomycin D (7-AAD), a fluorescent chemical compound with a strong affinity for DNA, was used (Paterson A M et al, 2011). FIG. 35B shows results from flow Cytometry experiments on live (7AAD negative) cells demonstrating the binding of mAb NMC-H103 (VH4/VK3) and NMC-C303 (solid black curve) to live OVCAR-3 (upper panel) and LL/2 cells (lower panel). In contrast, when OVCAR-3 and LL/2 cells were briefly treated with digestive enzyme Trypsin, while remaining alive (7AAD negative), the binding of mAb NMC-H103 (VH4/VK3) and NMC-C303 was reduced (Dashed line shifted back to that of background secondary Ab only), indicating loss of antibody binding epitope. These data demonstrate the specificity of these antibodies to their extracellularly accessible epitopes of the plasma membrane-bound M(H)DM2 on human ovarian cancer OVCAR-3 and mouse lung cancer LL/2 cells.

9.4 Example 16: Anti-Tumor Effect of Antibodies to the Plasma Membrane-Bound M(H)DM2 on Cancer Cells, which is in Part Dependent on Activation of the Immune System

Anti-cancer activity of humanized and chimeric antibodies to extracellularly accessible epitopes NMC-P1 and NMC-P3 of the plasma membrane-bound M(H)DM2 on cancer cells, was investigated utilizing NOD scid gamma mice (NSG: NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ). As described by the manufacturer (The Jackson Laboratory), these mice lack B-cell, T-cells, NK cells and complement and have defective dendritic cells and macrophages (Sultz L D et al, 2005). Utilization of NSG immune-deficient mice allows for engraftment of human peripheral monocytes (Hu-PBMSCs), which would enable testing of humanized and chimeric antibodies.

As shown in FIG. 36A, mouse colon cancer MC-38 cells were subcutaneously inoculated in NSG mice (n=12) and allowed to reach a tumor volume of 400 mm³ at which point they were randomly divided into 3 groups (n=4 mouse/group). Normal Hu-PBMCs (7×10⁶ PBMCs/mouse) were injected in tail vein of groups 1 and 3 mice, whereas mice in group 2 did not receive any Hu-PBMCs. A week later (tumors volume of 600 mm³), mice in group 1 received intraperitoneal injection of isotype control antibody (10 mg/kg), while mice in groups 2 and 3 received NMC-C303 (10 mg/kg). As shown in FIG. 36, mAb NMC-C303 was most effective in group 3 mice, which had PBMC (tumor volume of 1,492 mm³) as compared to mice in group 2, which received NMC-C303 but had received no Hu-PBMCs (tumor volume of 2,722 mm³). These observations demonstrate an immunological role for antibodies against plasma membrane-bound M(H)DM2 on cancer cells in suppression of tumor growth.

FIG. 36B demonstrates the immune-system's involvement in the anti-tumor activity of humanized NMC-H103 (VH4/VK3). As shown in FIG. 36B, human lung cancer A549 cells were subcutaneously injected into NSG mice (n=12) and were allowed to reach a tumor volume of 300 mm³ at which time mice were randomly divided into 3 groups (n=4 per group). Normal Hu-PBMCs (7×10⁶ PBMC/mouse) were injected in tail vein of mice in groups 1 and 3, whereas mice in group 2 did not receive any Hu-PBMCs. Mice in group 1 received 1 intraperitoneal injection of isotype control antibody (10 mg/kg), while mice in groups 2 and 3 received 1 intraperitoneal injection of NMC-H103 (VH4/VK3) (10 mg/kg). As shown in FIG. 36B, mice treated with 1 dose of mAb NMC-H103 (VH4/VK3) in presence of Hu-PBMCs had an average tumor volume of 784 mm³, while mice treated with 1 dose of mAb NMC-H103 (VH4/VK3) in the absence of Hu-PBMCs had an average of tumor volume of 1367 mm³, demonstrating that the anti-cancer activity of NMC-H103 (VH4/VK3) is significantly more effective in presence of Hu-PBMCs (FIG. 36B, black bar) than in the absence of Hu-PBMCs (FIG. 36B, middle bar), indicating an immune-assisted mechanism of action.

9.5 Example 17: Tumor Hyper-Progression Under Immunotherapy Results in an Increase in the Expression of Plasma Membrane-Bound M(H)DM2 on the Surface of the Hyper-Progressive Tumors

Recent observations in patients undergoing immune checkpoint inhibitor (ICI) treatment has identified a patient sub-population whose tumors hyper-progressed during or after treatment (Champiat, et al., Clin. Cancer Res. 23, 1920-1928 (2017)). Lung cancer M109 cells exhibit similar phenotype upon treatment with ICI (Shisuo Du et al., Oncoimmunology 2018; 7(4): e1408747).

FIG. 37A demonstrates the in vitro MTT cell proliferation assay of M109 cells treated with 75 ug/mL or 100 ug/mL of anti-PD1 antibody for 48 hours as described above. As shown in the bar graphs, a dose-dependent increase in cell proliferation is observed when cells are incubated with anti-PD1 antibody as compared to cells incubated with isotype control antibody at 100 ug/mL, demonstrating M109 cell proliferation in presence of anti-PD1 antibody.

FIG. 37B demonstrates the in vivo effect of anti-PD1 antibody on the growth of M109 tumor. NSG mice (n=8) were subcutaneously inoculated with M109 cells (50,000 cells/mouse). Two weeks later, mice were randomly divided in two groups. Mice in group 1 received isotype control antibody (12.5 mg/kg; xl/week), while mice in group 2 received anti-PD1 antibody (12.5 mg/kg; xl/week). As shown in FIG. 37B, after 3 weeks of treatment mice treated with anti-PD1 antibody (group 2) start to experience an increase in their tumor growth as compared to mice that received isotype control antibody (group 1), further confirming the results observed in vitro (FIG. 37A). Tumors from mice in both group 1 and 2 were harvested from sacrificed mice and assessed for the expression of plasma membrane-bound M(H)DM2.

FIG. 37C demonstrates results from flow cytometry experiments on freshly excised tumors from mice treated with isotype control or anti-PD1 antibodies (groups 1 and 2 discussed above). Single-cell suspension of tumors was performed as described above. Flow cytometry data has demonstrated more than 25% increase in the binding of NMC-H103 (VH4/VK3) and NMC-C303 to hyper-progressive tumors. As shown in FIG. 37C, an increased binding of NMC-H103 (VH4/VK3) was observed in hyper-progressive tumors that received anti-PD1 as compared to isotype control treated mice in group 1.

These observations demonstrate the correlation between the previously reported MDM2 gene amplification in hyper-progressive patients under immunotherapy (Kato et al., Clin. Cancer Res. 23, 4242-4250 (2017) and increased plasma membrane-bound MDM2 that could be targeted with antibodies to key accessible epitopes.

9.6 Example 18: Binding of Several Humanized mAb NMC-H103 Variants to NMC-P1

Example 38 presents data demonstrating the binding of multiple humanized mAb NMC-H103 variants to NMC-P1. In this Example, purified humanized variants of NMC-H103 antibody were tested by Biacore single cycle kinetic analysis for binding to NMC-P1, loading to a lower immobilization level (RL) of ˜3600 RU, the theoretical value to obtain an Rmax of about 75 RU to reduce the amount of antibody used. The surface was then allowed to stabilize. A three point, two-fold dilution range from 18.75 nM to 75 nM NMC-P.1 without regeneration between each concentration was used. The association phase for the three injections of increasing concentrations of NMC-P1 was monitored for 200 seconds each time and a single dissociation phase was measured for 200 seconds following the last injection. Regeneration of the Protein A surface was conducted using two injections of 10 mM glycine-HCL pH 1.5. Data was fitted using a 1-to-1 binding model. The sensorgrams and fitted data for the single cycle kinetics are shown in FIG. 38A and for multi-cycle kinetic analysis in FIG. 38B.

FIG. 38A presents the single cycle binding kinetics of humanized antibodies to NMC-P1, wherein the humanized antibodies comprise a combination of VH1, VH2, VH3, or VH4 humanized heavy chain variable region with VK 1, VK2, VK3, VK4, or VK5 humanized light chain variable region. FIG. 38B presents multi-cycle binding kinetics of humanized antibodies to NMC-P1, wherein the humanized antibodies comprise a combination of VH4, VH6, or VH7 humanized heavy chain variable region with VK3, VK6, or VK7 humanized light variable region. The sequences of these humanized heavy and light chains are provided in Tables 15-27 and Section 11 (see SEQ ID NOs: 287, 291, 295, 299, 305, and 309 for VH1, VH2, VH3, VH4, VH6, and VH7 sequences, respectively; and see SEQ ID Nos: 289, 293, 297, 301, 303, 307, 311, for VK1, VK2, VK3, VK4, VK5, VK6, and VK7 sequences, respectively).

9.7 Conclusions Based on Examples 13-18

In Examples 1-12, key segments of plasma membrane M(H)DM2 (PM-HDM2) that are extracellularly available for drug targeting were identified. Three examples of such extracellularly available regions of membrane-bound M(H)DM2 are: NMC-P1, NMC-P2 and NMC-P3 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3). Mouse monoclonal antibodies were developed against these key epitopes and their binding to a variety of cancer cells and their lack of binding or significantly lower binding ability to untransformed cells was demonstrated. In addition, it was demonstrated that not all antibodies to M(H)DM2 are able to bind to plasma membrane-bound HDM2. It was also demonstrated that select antibodies against plasma membrane-bound HDM2 that bind to extra-cellularly accessible epitopes have anti-cancer activity in vitro by various mechanisms including CDC and ADCC. Moreover, it was shown that select antibodies against extra-cellularly accessible regions of plasma membrane-bound HDM2 demonstrate in vivo anti-tumor activity in mouse models including but not limited to lung, colon, and pancreatic cancers.

In Examples 9-13, humanized and chimeric monoclonal antibodies were developed against these key epitopes and their binding to intact, un-permeabilized cancer cells was demonstrated to be similar to that of plasma membrane markers (FIG. 33). It was also shown that these antibodies are specific for M(H)DM2 and it was shown that their binding to intact, un-permeabilized cancer cells can be competed with their epitope-specific peptides (FIG. 35A).

In addition, it was shown that the tested humanized and chimeric antibodies against extracellularly accessible epitopes of plasma membrane-bound HDM2 have anti-tumor effect, and the anti-tumor effect of these antibodies is in part immune-mediated, as their in-vivo efficacy is reduced in in the immune-deficient NSG mice (FIGS. 36A and 36B).

Immune checkpoint inhibitors (ICIs) such as anti-PD-1/PDL-1 and CTLA-4 have demonstrated long lasting effects in various cancers and have become standard of care for some patients. However, recent observations have identified a sub-population of patients who experienced accelerated tumor progression under ICI (Champiat et al., Clin. Cancer Res. 23, 1920-1928 (2017); Kato et al., Clin. Cancer Res. 23, 4242-4250 (2017); Saâda-Bouzid et al., Ann. Oncol. 28, 1605-1611 (2017); Ferrara et al., JAMA Oncol. (2018); and Zuazo-Ibarra et al., 2018). According to these observations, hyperprogressive disease was not limited to any particular cancer type. Hyper-progression under ICI was observed in non-small cell lung cancer (NSCLC) with an incident ranging from 8-21% (Kato et al., Clin. Cancer Res. 23, 4242-4250 (2017); Ferrara et al., JAMA Oncol. (2018); and Zuazo-Ibarra et al., (2018)), in melanoma with an incident of 9% (Champiat et al., Clin. Cancer Res. 23, 1920-1928 (2017)) and poor median overall survivals (<3 months) was observed in patients with hyperprogressive disease (Champiat et al., Clin. Cancer Res. 23, 1920-1928 (2017); and Ferrara et al., JAMA Oncol. (2018)). These observations were supported by Phase III clinical trials involving patients with NSCLC (Borghaei et al., N. Engl. J. Med. 373, 1627-1639 (2015)), HNSCC (Ferris et al., N. Engl. J. Med. 375, 1856-1867 (2016)) and Urethrial carcinoma (Bellmunt et al,. N. Engl. J. Med. 376, 1015-1026 (2017)). MDM2/4 gene amplification and EGFR alterations were found to be associated with time to treatment failure (TTF) (Kato et al., Clin. Cancer Res. 23, 4242-4250 (2017)). In two studies, around two third of the patients with MDM2/MDM4 gene amplification were found to be hyperprogressors (Kato et al., Clin. Cancer Res. 23, 4242-4250 (2017) A. K. Singavi et al., Ann Oncol 2017; 28).

A mouse model of lung cancer (M109) has demonstrated phenotypic similarities to that of hyperprogressive patients under ICI (Shisuo Oncoimmunology. 2018; 7(4): e1408747). Data presented here demonstrates an increase in the binding of antibodies to extracellularly accessible regions of plasma membrane-bound M(H)DM2 of ICI-treated hyper-progressive tumors as compared to untreated tumors (FIG. 37). Therefore, data presented here demonstrates a correlation between MDM2 gene amplification and an increase in the expression of plasma membrane-bound M(H)DM2. Taken together, data presented herein demonstrate the rationale for using antibodies to plasma membrane-bound M(H)DM2 in combination with ICI (before, during or after administraton of ICI).

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11. SEOUENCES (NMC-P1) SEQ ID NO: 1 MCNTNMSVPTDGAVT (NMC-P2) SEQ ID NO: 2 TTSQIPASEQE (NMC-P3) SEQ ID NO: 3 CPVCRQPIQMIVLTYFP (Human HDM2 Protein): SEQ ID NO: 4 MCNTNMSVPT DGAVTTSQIP ASEQETLVRP KPLLLKLLKS VGAQKDTYTM KEVLFYLGQY IMTKRLYDEK QQHIVYCSND LLGDLFGVPS FSVKEHRKIY TMIYRNLVVV NQQESSDSGT SVSENRCHLE GGSDQKDLVQ ELQEEKPSSS HLVSRPSTSS RRRAISETEE NSDELSGERQ RKRHKSDSIS LSFDESLALC VIREICCERS SSSESTGTPS NPDLDAGVSE HSGDWLDQDS VSDQFSVEFE VESLDSEDYS LSEEGQELSD EDDEVYQVTV YQAGESDTDS FEEDPEISLA DYWKCTSCNE MNPPLPSHCN RCWALRENWL PEDKGKDKGE ISEKAKLENS TQAEEGFDVP DCKKTIVNDS RESCVEENDD KITQASQSQE SEDYSQPSTS SSIIYSSQED VKEFEREETQ DKEESVESSL PLNAIEPCVI CQGRPKNGCI VHGKTGHLMA CFTCAKKLKK RNKPCPVCRQ PIQMIVLYF P (Mouse MDM2 Protein): SEQ ID NO: 5 MCNTNMSVST EGAASTSQIP ASEQETLVRP KPLLLKLLKS VGAQNDTYTM KEIIFYIGQY IMTKRLYDEK QQHIVYCSND LLGDVFGVPS FSVKEHRKIY AMIYRNLVAV SQQDSGTSLS ESRRQPEGGS DLKDPLQAPP EEKPSSSDLI SRLSTSSRRR SISETEENTD ELPGERHRKR RRSLSFDPSL GLCELREMCS GGSSSSSSSS SESTETPSHQ DLDDGVSEHS GDCLDQDSVS DQFSVEFEVE SLDSEDYSLS DEGHELSDED DEVYRVTVYQ TGESDTDSFE GDPEISLADY WKCTSCNEMN PPLPSHCKRC WTLRENWLPD DKGKDKVEIS EKAKLENSAQ AEEGLDVPDG KKLTENDAKE PCAEEDSEEK AEQTPLSQES DDYSQPSTSS SIVYSSQESV KELKEETQDK DESVESSFSL NAIEPCVICQ GRPKNGCIVH GKTGHLMSCF TCAKKLKKRN KPCPVCRQPI QMIVLTYFN (Human HDM4 protein): SEQ ID NO: 6 MISFSTSAQCSTSDSACRISPGQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQ YIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLATATTDAAQT LALAQDHSMDIPSQDQLKQSAEESSTSRKRTTEDDIPTLPTSEHKCIHSREDEDLIEN LAQDETSRLDLGFEEWDVAGLPWWFLGNLRSNYTPRSNGSTDLQTNQDVGTAIVSDTT DDLWFLNESVSEQLGVGIKVEAADTEQTSEEVGKVSDKKVIEVGKNDDLEDSKSLSDD TDVEVTSEDEWQCTECKKFNSPSKRYCFRCWALRKDWYSDCSKLTHSLSTSDITAIPE KENEGNDVPDCRRTISAPVVRPKDAYIKKENSKLFDPCNSVEFLDLAHSSESQETISS MGEQLDNLSEQRTDTENMEDCQNLLKPCSLCEKRPRDGNIIHGRTGHLVTCFHCARRL KKAGASCPICKKE IQLVIKVFIA (Mouse MDMX-S protein): SEQ ID NO: 7 MTSHSTSAQCSASDSACRISSEQISQVRPKLQLLKILHAAGAQGEVFTMKEVMHYLGQYIMVK QLYDQQEQHMVYCGGDLLGDLLGCQSFSVKDPSPLYDMLRKNLVTSASINTARC NRILQSQKKN M(H)DM2/4 variants: (HDM2 variant MDM2-A which lacks amino acids residues 28-222 of SEQ ID NO: 4 as indicated in the below sequence by “(28-222)”): SEQ ID NO: 8 MCNTNMSVPTDGAVTTSQIPASEQETLD (28-222) LDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAG ESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKA KLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYS SQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKK LKKRNKPCPVCRQPIQMIVLTYFP (HDM2 variant MDM2-A1 which lacks amino acids residues 28-222  and 275-300 of SEQ ID NO: 4 as indicated in the below sequence  by “(28-222)” and “(275-300)”): SEQ ID NO: 9 MCNTNMSVPT DGAVTTSQIP ASEQETLD (28-222) LDAGVSEH SGDWLDQDSV SDQFSVEFEV ESLDSEDYSL SEEGQELSDE DDEDY (275-300) WKCTS CNEMNPPLPS HCNRCWALRE NWLPEDKGKD KGEISEKAKL ENSTQAEEGF DVPDCKKTIV NDSRESCVEE NDDKITQASQ SQESEDYSQP STSSSIIYSS QEDVKEFERE ETQDKEESVE SSLPLNAIEP CVICQGRPKN GCIVHGKTGH LMACFTCAKK LKKRNKPCPV CRQPIQMIVL TYFP (HDM2 variant MDM2-B which lacks amino acids residues 28-300  of SEQ ID NO: 4 as indicated in the below sequence by “(28-300)”): SEQ ID NO: 10 MCNTNMSVPTDGAVTTSQIPASEQETLD (28-300) YWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKK TIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVE SSLPLNAIEPCVICQGRPKNGCIVHG KTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (HDM2 variant MDM2-C which lacks amino acids residues 53-222 of  SEQ ID NO: 4 as indicated in the below sequence by “(53-222)”): SEQ ID NO: 11 MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKED (53-222) LDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESD TDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENS TQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQED VKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKK RNKPCPVCRQPIQMIVLTYFP (HDM2 variant MDM2-D which lacks amino acids residues 30-388 of SEQ ID NO: 4 as indicated in the below sequence by “(30-388)”): SEQ ID NO: 12 MCNTNMSVPTDGAVTTSQIPASEQETLVRQ (30-388) ESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHG KTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (HDM2 variant MDM2-E which lacks amino acids residues 76- 102 and 103-491 of SEQ ID NO: 4 as indicated in the below sequence  by “(76-102)” and “(103-491)”): SEQ ID NO: 13 MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLF YLGQY IMTKRLYDEKQQHIVN (76-102) (103-491) DCANLFPLVDLSIRELYISNYITLGI (HDM2 variant MDM2-F which lacks amino acids residues 53-97 of SEQ ID NO: 4 as indicated in the below sequence by “(53-97)”): SEQ ID NO: 14 MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKE (53-97) KIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPS TSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTG TPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQ VTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKD KGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQP STSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGH LMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (HDM2 variant MDM2-G which lacks amino acids residues 115-169 of SEQ ID NO: 4 as indicated in the below sequence by “(115-169)”): SEQ ID NO: 15 MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQY IMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQEE (115- 169) NSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHS GDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEED PEISLADYWKCTSNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEE GFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFE REETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPC PVCRQPIQMIVLTYFP (HDM2 variant MDM2-11): SEQ ID NO: 16 MVRSRQMCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVL FYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQE SSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDE LSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGD WLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEED PEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAE EGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEF EREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKP CPVCRQPIQMIVLTYFP (HDM2 variant MDM2-KB2 which lacks amino acids residues 157-248 of SEQ ID NO: 4 as indicated in the below sequence by “(157-248)”): SEQ ID NO: 17 MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEDYWKCTSC NEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVN DSRESCVEENDDKITQASQSQESEDYSQPSTSSSII (157- 248) YSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACF TCAKKLKKRNKPCPVCRQPIQMIVLTYFP

TABLE 4 mAb NMC-103 Heavy Chain CDR and HFR Sequences using Chothia, AbM, Kabat, Contact and IMGT CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹ Length NO: HFR1 Chothia EVQLQESGGGLVQPGGSLRLSCTTS-----  1-25 25 72 AbM EVQLQESGGGLVQPGGSLRLSCTTS-----  1-25 25 72 Kabat EVQLQESGGGLVQPGGSLRLSCTTSGFTFT  1-30 30 73 Contact EVQLQESGGGLVQPGGSLRLSCTTSGFTF-  1-29 29 74 CDR-H1 Chothia GFTFTHY--- 26-32 7 18 AbM GFTFTHYYMS 26-35 10 42 Kabat -----HYYMS 31-35 5 43 Contact ----THYYMS 30-35 6 44 IMGT -FTFTHYY-- 27-33 7 144 HFR2 Chothia YMSWVRQPPGKALEWLGFI 33-51 19 75 AbM ---WVRQPPGKALEWLG-- 36-49 14 76 Kabat ---WVRQPPGKALEWLG-- 36-49 14 76 Contact ---WVRQPPGKALE----- 36-46 11 77 CDR-H2 Chothia -----RNKAKGYT--------- 52-59 8 19 AbM ---FIRNKAKGYTAE------- 50-61 12 45 Kabat ---FIRNKAKGYTAEYSASVKG 50-68 19 46 Contact WLGFIRNKAKGYTAE------- 47-61 15 47 IMGT ----IRNKAKGYTA-------- 51-60 10 145 HFR3 Chothia AEYSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR  60-100 41 78 AbM --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR  62-100 39 79 Kabat ---------RFTISRDNSQSILYLQMNTLRPEDSATYYCAR  69-100 32 80 Contact --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYC-- 62-98 37 81 CDR-H3 Chothia --DIGDN 101-105 5 20 AbM --DIGDN 101-105 5 20 Kabat --DIGDN 101-105 5 20 Contact ARDIGD-  99-104 6 48 IMGT ARDIGDN  99-105 7 146 HFR4 Chothia -WGQGTLVTVSS 106-116 11 82 AbM -WGQGTLVTVSS 106-116 11 82 Kabat -WGQGTLVTVSS 106-116 11 82 Contact NWGQGTLVTVSS 105-116 12 83 ¹The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 36) of NMC-103.

TABLE 5 mAb NMC-103 Light Chain CDR and HFR Sequences using Chothia, AbM, Kabat, Contact and IMGT CDR definitions: SEQ ID Region Definition Sequence Fragment Residues² Length NO: LFR1 Chothia DIVMTQAAFSNPVTLGTSASISC------  1-23 23 84 AbM DIVMTQAAFSNPVTLGTSASISC------  1-23 23 84 Kabat DIVMTQAAFSNPVTLGTSASISC------  1-23 23 84 Contact DIVMTQAAFSNPVTLGTSASISCRSSKNL  1-29 29 85 CDR-L1 Chothia RSSKNLLHSNGITYLY-- 24-39 16 21 AbM RSSKNLLHSNGITYLY-- 24-39 16 21 Kabat RSSKNLLHSNGITYLY-- 24-39 16 21 Contact ------LHSNGITYLYWY 30-41 12 49 IMGT ---KNLLHSNGITY---- 27-37 11 147 LFR2 Chothia WYLQRPGQSPQLLIS 40-54 15 86 AbM WYLQRPGQSPQLLIS 40-54 15 86 Kabat WYLQRPGQSPQLLIS 40-54 15 86 Contact --LQRPGQSPQ---- 42-50 9 87 CDR-L2 Chothia ----RVSNLAS 55-61 7 22 AbM ----RVSNLAS 55-61 7 22 Kabat ----RVSNLAS 55-61 7 22 Contact LLISRVSNLA- 51-60 10 50 IMGT ----RVS---- 55-57 3 LFR3 Chothia -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 32 88 AbM -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 32 88 Kabat -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 62-93 32 88 Contact SGVPNRFSGSESGTDFTLRISRVEAEDVGVYFC 61-93 33 89 CDR-L3 Chothia AQLLELPYT  94-102 9 23 AbM AQLLELPYT  94-102 9 23 Kabat AQLLELPYT  94-102 9 23 Contact AQLLELPY-  94-101 8 51 IMGT AQLLELPYT  94-102 9 23 LFR4 Chothia -FGGGTKLEIK 103-112 10 90 AbM -FGGGTKLEIK 103-112 10 90 Kabat -FGGGTKLEIK 103-112 10 90 Contact TFGGGTKLEIK 102-112 11 91 ²The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 37) of NMC-103.

TABLE 6 mAb NMC-204 Heavy Chain CDR and HFR Sequences using Chothia, AbM, Kabat, Contact and IMGT CDR defintions: SEQ ID Region Definition Sequence Fragment Residues³ Length NO: HFR1 Chothia EVQLQESGSVLVRPGASVKLSCKAS-----  1-25 25 92 AbM EVQLQESGSVLVRPGASVKLSCKAS-----  1-25 25 92 Kabat EVQLQESGSVLVRPGASVKLSCKASGDTLS  1-30 30 93 Contact EVQLQESGSVLVRPGASVKLSCKASGDTL-  1-29 29 94 CDR-H1 Chothia GDTLSGS--- 26-32 7 24 AbM GDTLSGSWMH 26-35 10 52 Kabat -----GSWMH 31-35 5 53 Contact ----SGSWMH 30-35 6 54 IMGT GDTLSGSW-- 26-33 8 148 HFR2 Chothia WMHWAMQRPGQGLEWIGEI 33-51 19 95 AbM ---WAMQRPGQGLEWIG-- 36-49 14 96 Kabat ---WAMQRPGQGLEWIG-- 36-49 14 96 Contact ---WAMQRPGQGLE----- 36-46 11 97 CDR-H2 Chothia -----HLNRGT--------- 52-57 6 25 AbM ---EIHLNRGTTN------- 50-59 10 55 Kabat ---EIHLNRGTTNYNEKFKG 50-66 17 56 Contact WIGEIHLNRGTTN------- 47-59 13 57 IMGT ----IHLNRGTT-------- 51-58 8 143 HFR3 Chothia TNYNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 58-98 41 98 AbM --YNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 60-98 39 99 Kabat ---------KATVTVDTSSSTAYVDLSSLTSEDSAVYYCAR 67-98 32 100 Contact --YNEKFKGKATVTVDTSSSTAYVDLSSLTSEDSAVYYC-- 60-96 37 101 CDR-H3 Chothia --SPGFAY  99-104 6 26 AbM --SPGFAY  99-104 6 26 Kabat --SPGFAY  99-104 6 26 Contact ARSPGFA-  97-103 7 58 IMGT ARSPGFA-  97-103 7 58 HFR4 Chothia -WGQGTLVTVSA 105-115 11 102 AbM -WGQGTLVTVSA 105-115 11 102 Kabat -WGQGTLVTVSA 105-115 11 102 Contact YWGQGTLVTVSA 104-115 12 103 ³The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 38) of NMC-204.

TABLE 7 mAb NMC-204 Light Chain CDR and HFR Sequences using Chothia, AbM, Kabat, Contact and IMGT CDR defintions SEQ ID Region Definition Sequence Fragment Residues⁴ Length NO: LFR1 Chothia GIVMTQAAPSVPVTPGESVSISC------  1-23 23 104 AbM GIVMTQAAPSVPVTPGESVSISC------  1-23 23 104 Kabat GIVMTQAAPSVPVTPGESVSISC------  1-23 23 104 Contact GIVMTQAAPSVPVTPGESVSISCRSSKSL  1-29 29 105 CDR-L1 Chothia RSSKSLLHSNGNSYLY-- 24-39 16 27 AbM RSSKSLLHSNGNSYLY-- 24-39 16 27 Kabat RSSKSLLHSNGNSYLY-- 24-39 16 27 Contact ------LHSNGNSYLYWF 30-41 12 59 IMGT ---KSLLHSNGNSY---- 27-37 11 141 LFR2 Chothia WFLQRPGQSPQLLIY 40-54 15 106 AbM WFLQRPGQSPQLLIY 40-54 15 106 Kabat WFLQRPGQSPQLLIY 40-54 15 106 Contact --LQRPGQSPQ---- 42-50 9 107 CDR-L2 Chothia ----RMSNLAS 55-61 7 28 AbM ----RMSNLAS 55-61 7 28 Kabat ----RMSNLAS 55-61 7 28 Contact LLIYRMSNLA- 51-60 10 60 IMGT ----RMS---- 55-57 3 LFR3 Chothia -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62-93 32 108 AbM -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62-93 32 108 Kabat -GVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 62-93 32 108 Contact SGVPDRFSGSGSGTAFTLRITRVEAEDVGVYYC 61-93 33 109 CDR-L3 Chothia MQHLEYPFT  94-102 9 29 AbM MQHLEYPFT  94-102 9 29 Kabat MQHLEYPFT  94-102 9 29 Contact MQHLEYPF-  94-101 8 61 IMGT MQHLEYPFT  94-102 9 29 LFR4 Chothia -FGSGTKLEIK 103-112 10 110 AbM -FGSGTKLEIK 103-112 10 110 Kabat -FGSGTKLEIK 103-112 10 110 Contact TFGSGTKLEIK 102-112 11 111 ⁴The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 39) of NMC-204.

TABLE 8 mAb NMC-303 Heavy Chain CDR and HFR Sequences using Chothia, AbM, Kabat and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues⁵ Length NO: HFR1 Chothia QVQLQQPGAELVKPGASVKLSCKAS-----  1-25 25 112 AbM QVQLQQPGAELVKPGASVKLSCKAS-----  1-25 25 112 Kabat QVQLQQPGAELVKPGASVKLSCKASGYTFT  1-30 30 113 Contact QVQLQQPGAELVKPGASVKLSCKASGYTF-  1-29 29 114 CDR-H1 Chothia GYTFTSY--- 26-32 7 30 AbM GYTFTSYYMY 26-35 10 62 Kabat -----SYYMY 31-35 5 63 Contact ----TSYYMY 30-35 6 64 HFR2 Chothia YMYWVKQRPGQGLEWIGGI 33-51 19 115 AbM ---WVKQRPGQGLEWIG-- 36-49 14 116 Kabat ---WVKQRPGQGLEWIG-- 36-49 14 116 Contact ---WVKQRPGQGLE----- 36-46 11 117 CDR-H2 Chothia -----NPRNGG--------- 52-57 6 31 AbM ---GINPRNGGTN------- 50-59 10 65 Kabat ---GINPRNGGTNFNEKFKN 50-66 17 66 Contact WIGGINPRNGGTN------- 47-59 13 67 HFR3 Chothia TNFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 58-98 41 118 AbM --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 60-98 39 119 Kabat ---------KATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 67-98 32 120 Contact --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYC-- 60-96 37 121 CDR-H3 --SGYYAMDY  99-106 8 32 AbM --SGYYAMDY  99-106 8 32 Kabat --SGYYAMDY  99-106 8 32 Contact TRSGYYAMD-  97-105 9 68 HFR4 Chothia -WGQGTSVTVSS 107-117 11 122 AbM -WGQGTSVTVSS 107-117 11 122 Kabat -WGQGTSVTVSS 107-117 11 122 Contact YWGQGTSVTVSS 106-117 12 123 ⁵The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 40) of NMC-303.

TABLE 9 mAb NMC-303 Light Chain CDR and HFR Sequences using  Chothia, AbM, Kabat and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues⁶ Length NO: LFR1 Chothia DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 AbM DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 Kabat DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 Contact DIQMTQTTSSLSASLGDRVTISCRASQDI  1-29 29 125 CDR-L1 Chothia RASQDISNFLN-- 24-34 11 33 AbM RASQDISNFLN-- 24-34 11 33 Kabat RASQDISNFLN-- 24-34 11 33 Contact ------SNFLNWY 30-36 7 69 LFR2 Chothia WYQQKPDGTVKLLIY 35-49 15 126 AbM WYQQKPDGTVKLLIY 35-49 15 126 Kabat WYQQKPDGTVKLLIY 35-49 15 126 Contact --QQKPDGTVK---- 37-45 9 127 CDR-L2 Chothia ----YTSRLHS 50-56 7 34 AbM ----YTSRLHS 50-56 7 34 Kabat ----YTSRLHS 50-56 7 34 Contact LLIYYTSRLH- 46-55 10 70 LFR3 Chothia -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 AbM -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 Kabat -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 Contact SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 56-88 33 129 CDR-L3 Chothia QQGNTLPRT 89-97 9 35 AbM QQGNTLPRT 89-97 9 35 Kabat QQGNTLPRT 89-97 9 35 Contact QQGNTLPR- 89-96 8 71 LFR4 Chothia -FGGGTKLEIK  98-107 10 130 AbM -FGGGTKLEIK  98-107 10 130 Kabat -FGGGTKLEIK  98-107 10 130 Contact TFGGGTKLEIK  97-107 11 131 ⁶The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 41) of NMC-303.

TABLE 11 mAb NMC-C303 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues⁷ Length NO: HFR1 Chothia QVQLQQPGAELVKPGASVKLSCKAS-----  1-25 25 112 AbM QVQLQQPGAELVKPGASVKLSCKAS-----  1-25 25 112 Kabat QVQLQQPGAELVKPGASVKLSCKASGYTFT  1-30 30 113 Contact QVQLQQPGAELVKPGASVKLSCKASGYTF-  1-29 29 114 CDR-H1 Chothia GYTFTSY--- 26-32 7 30 AbM GYTFTSYYMY 26-35 10 62 Kabat -----SYYMY 31-35 5 63 Contact ----TSYYMY 30-35 6 64 HFR2 Chothia YMYWVKQRPGQGLEWIGGI 33-51 19 115 AbM ---WVKQRPGQGLEWIG-- 36-49 14 116 Kabat ---WVKQRPGQGLEWIG-- 36-49 14 116 Contact ---WVKQRPGQGLE----- 36-46 11 117 CDR-H2 Chothia -----NPRNGG--------- 52-57 6 31 AbM ---GINPRNGGTN------- 50-59 10 65 Kabat ---GINPRNGGTNFNEKFKN 50-66 17 66 Contact WIGGINPRNGGTN------- 47-59 13 67 HFR3 Chothia TNFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 58-98 41 118 AbM --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 60-98 39 119 Kabat ---------KATLTADKSSTTAYMQLSSLTSEDSAVYYCTR 67-98 32 120 Contact --FNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYC-- 60-96 37 121 CDR-H3 Chothia --SGYYAMDY  99-106 8 32 AbM --SGYYAMDY  99-106 8 32 Kabat --SGYYAMDY  99-106 8 32 Contact TRSGYYAMD-  97-105 9 68 HFR4 Chothia -WGQGTSVTVSS 107-117 11 122 AbM -WGQGTSVTVSS 107-117 11 122 Kabat -WGQGTSVTVSS 107-117 11 122 Contact YWGQGTSVTVSS 106-117 12 123 ⁷⁷The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 40) of NMC-C303.

TABLE 12 mAb NMC-C303 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues⁸ Length NO: LFR1 Chothia DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 AbM DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 Kabat DIQMTQTTSSLSASLGDRVTISC------  1-23 23 124 Contact DIQMTQTTSSLSASLGDRVTISCRASQDI  1-29 29 125 CDR-L1 Chothia RASQDISNFLN-- 24-34 11 33 AbM RASQDISNFLN-- 24-34 11 33 Kabat RASQDISNFLN-- 24-34 11 33 Contact ------SNFLNWY 30-36 7 69 LFR2 Chothia WYQQKPDGTVKLLIY 35-49 15 126 AbM WYQQKPDGTVKLLIY 35-49 15 126 Kabat WYQQKPDGTVKLLIY 35-49 15 126 Contact --QQKPDGTVK---- 37-45 9 127 CDR-L2 Chothia ----YTSRLHS 50-56 7 34 AbM ----YTSRLHS 50-56 7 34 Kabat ----YTSRLHS 50-56 7 34 Contact LLIYYTSRLH- 46-55 10 70 LFR3 Chothia -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 AbM -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 Kabat -GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 57-88 32 128 Contact SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC 56-88 33 129 CDR-L3 Chothia QQGNTLPRT 89-97 9 35 AbM QQGNTLPRT 89-97 9 35 Kabat QQGNTLPRT 89-97 9 35 Contact QQGNTLPR- 89-96 8 71 LFR4 Chothia -FGGGTKLEIK  98-107 10 130 AbM -FGGGTKLEIK  98-107 10 130 Kabat -FGGGTKLEIK  98-107 10 130 Contact TFGGGTKLEIK  97-107 11 131 ⁸⁸The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 41) of NMC-C303.

TABLE 13 mAb NMC-C103.VH0 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues⁹ Length NO: HFR1 Chothia EVKLVESGGGLVQPGGSLRLSCTTS-----  1-25 25 149 AbM EVKLVESGGGLVQPGGSLRLSCTTS-----  1-25 25 149 Kabat EVKLVESGGGLVQPGGSLRLSCTTSGFTFT  1-30 30 150 Contact EVKLVESGGGLVQPGGSLRLSCTTSGFTF-  1-29 29 151 CDR-H1 Chothia GFTFTHY--- 26-32 7 18 AbM GFTFTHYYMS 26-35 10 42 Kabat -HYYMS---- 31-35 5 43 Contact THYYMS---- 30-35 6 44 HFR2 Chothia YMSWVRQPPGKALEWLGFI 33-51 19 75 AbM ---WVRQPPGKALEWLG-- 36-49 14 76 Kabat ---WVRQPPGKALEWLG-- 36-49 14 76 Contact ---WVRQPPGKALE----- 36-46 11 77 CDR-H2 Chothia -----RNKAKGYT--------- 52-59 8 19 AbM ---FIRNKAKGYTAE------- 50-61 12 45 Kabat ---FIRNKAKGYTAEYSASVKG 50-68 19 46 Contact WLGFIRNKAKGYTAE------- 47-61 15 47 HFR3 Chothia AEYSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR  60-100 41 78 AbM --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYCAR  62-100 39 79 Kabat ---------RFTISRDNSQSILYLQMNTLRPEDSATYYCAR  69-100 32 80 Contact --YSASVKGRFTISRDNSQSILYLQMNTLRPEDSATYYC-- 62-98 37 81 CDR-H3 Chothia --DIGDN 101-105 5 20 AbM --DIGDN 101-105 5 20 Kabat --DIGDN 101-105 5 20 Contact ARDIGD-  99-104 6 48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 82 AbM -WGQGTLVTVSS 106-116 11 82 Kabat -WGQGTLVTVSS 106-116 11 82 Contact NWGQGTLVTVSS 105-116 12 83 ⁹The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 283) of NMC-C103.VH0.

TABLE 14 mAb NMC-C103.VK0 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁰ Length NO: LFR1 Chothia DIVMTQAAFSNPVTLGTSASISC------   1-23 23 84 AbM DIVMTQAAFSNPVTLGTSASISC------   1-23 23 84 Kabat DIVMTQAAFSNPVTLGTSASISC------   1-23 23 84 Contact DIVMTQAAFSNPVTLGTSASISCRSSKNL   1-29 29 85 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16 21 AbM RSSKNLLHSNGITYLY--  24-39 16 21 Kabat RSSKNLLHSNGITYLY--  24-39 16 21 Contact ------LHSNGITYLYWY  30-41 12 49 LFR2 Chothia WYLQRPGQSPQLLIS  40-54 15 86 AbM WYLQRPGQSPQLLIS  40-54 15 86 Kabat WYLQRPGQSPQLLIS  40-54 15 86 Contact --LQRPGQSPQ----  42-50  9 87 CDR-L2 Chothia ----RVSNLAS  55-61  7 22 AbM ----RVSNLAS  55-61  7 22 Kabat ----RVSNLAS  55-61  7 22 Contact LLISRVSNLA-  51-60 10 50 LFR3 Chothia -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC  62-93 32 88 AbM -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC  62-93 32 88 Kabat -GVPNRFSGSESGTDFTLRISRVEAEDVGVYFC  62-93 32 88 Contact SGVPNRFSGSESGTDFTLRISRVEAEDVGVYFC  62-93 33 89 CDR-L3 Chothia AQLLELPYT  94-102  9 23 AbM AQLLELPYT  94-102  9 23 Kabat AQLLELPYT  94-102  9 23 Contact AQLLELPY-  94-102  8 51 LFR4 Chothia -FGGGTKLEIK 103-112 10 90 AbM -FGGGTKLEIK 103-112 10 90 Kabat -FGGGTKLEIK 103-112 10 90 Contact TFGGGTKLEIK 102-112 11 91 ¹⁰The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 285) of NMC-C103.VK0.

TABLE 15 mAb NMC-H103.VH1 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat,and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹¹ Length NO: HFR1 Chothia EVKLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 152 AbM EVKLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 152 Kabat EVKLVESGGGLVQPGPSLRLSCTTSGFTFT   1-30 30 153 Contact EVKLVESGGGLVQPGPSLRLSCTTSGFTF-   1-29 29 154 CDR-H1 Chothia GFTFTHY---  26-32  7  18 AbM GFTFTHYYMS  26-35 10  42 Kabat -----HYYMS  31-35  5  43 Contact ----THYYMS  30-35  6  44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 155 AbM ---WVRQAPGKGLEWLG--  36-49 14 156 Kabat ---WVRQAPGKGLEWLG--  36-49 14 156 Contact ---WVRQAPGKGLE-----  36-46 11 157 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8  19 AbM ---FIRNKAKGYTAE-------  50-61 12  45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19  46 Contact WLGFIRNKAKGYTAE-------  47-61 15  47 HFR3 Chothia AEYSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYCAR  60-100 41 158 AbM --YSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYCAR  62-100 39 159 Kabat ---------RFTISRDNSKSTLYLQMNTLRAEDSATYYCAR  69-100 32 160 Contact --YSASVKGRFTISRDNSKSTLYLQMNTLRAEDSATYYC--  62-98 37 161 CDR-H3 Chothia --DIGDN 101-105  5  20 AbM --DIGDN 101-105  5  20 Kabat --DIGDN 101-105  5  20 Contact ARDIGD-  99-104  6  48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 162 AbM -WGQGTLVTVSS 106-116 11 162 Kabat -WGQGTLVTVSS 106-116 11 162 Contact NWGQGTLVTVSS 105-116 12 163 ¹¹The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 287) of NMC-H103.VH1.

TABLE 16 mAb NMC-H103.VK1 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹² Length NO: LFR1 Chothia DIVMTQAAFSNPVTLGQPASISC------   1-23 23 164 AbM DIVMTQAAFSNPVTLGQPASISC------   1-23 23 164 Kabat DIVMTQAAFSNPVTLGQPASISC------   1-23 23 164 Contact DIVMTQAAFSNPVTLGQPASISCRSSKNL   1-29 29 165 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16  21 AbM RSSKNLLHSNGITYLY--  24-39 16  21 Kabat RSSKNLLHSNGITYLY--  24-39 16  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 166 AbM WYLQKPGQSPQLLIS  40-54 15 166 Kabat WYLQKPGQSPQLLIS  40-54 15 166 Contact --LQKPGQSPQ----  42-50  9 167 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61  7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 168 AbM -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 168 Kabat -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 168 Contact SGVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 33 169 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 170 AbM -FGGGTKVEIK 103-112 10 170 Kabat -FGGGTKVEIK 103-112 10 170 Contact TFGGGTKVEIK 102-112 11 171 ¹²The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 289) of NMC-H103.VK1.

TABLE 17 mAb NMC-H103.VH2 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat,and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹³ Length NO: HFR1 Chothia EVKLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 172 AbM EVKLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 172 Kabat EVKLVESGGGLVQPGPSLRLSCTTSGFTFT   1-30 30 173 Contact EVKLVESGGGLVQPGPSLRLSCTTSGFTF-   1-29 29 174 CDR-H1 Chothia GFTFTHY---  26-32  7  18 AbM GFTFTHYYMS  26-35 10  42 Kabat -----HYYMS  31-35  5  46 Contact ----THYYMS  30-35  6  44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 175 AbM ---WVRQAPGKGLEWLG--  36-49 14 176 Kabat ---WVRQAPGKGLEWLG--  36-49 14 176 Contact ---WVRQAPGKGLE-----  36-46 11 177 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8  19 AbM ---FIRNKAKGYTAE-------  50-61 12  45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19  46 Contact WLGFIRNKAKGYTAE-------  47-61 15  47 HFR3 Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  60-100 41 178 AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  62-100 39 179 Kabat ---------RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  69-100 32 180 Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYC--  62-98 37 181 CDR-H3 Chothia --DIGDN 101-105  5  20 AbM --DIGDN 101-105  5  20 Kabat --DIGDN 101-105  5  20 Contact ARDIGD-  99-104  6  48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 182 AbM -WGQGTLVTVSS 106-116 11 182 Kabat -WGQGTLVTVSS 106-116 11 182 Contact NWGQGTLVTVSS 105-116 12 183 ¹³The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 291) of NMC-H103.VH2.

TABLE 18 mAb NMC-H103.VK2 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁴ Length NO: LFR1 Chothia DIVMTQTPLSSPVTLGQPASISC-----   1-23 23 184 AbM DIVMTQTPLSSPVTLGQPASISC-----   1-23 23 184 Kabat DIVMTQTPLSSPVTLGQPASISC-----   1-23 23 184 Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL   1-29 29 185 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16  21 AbM RSSKNLLHSNGITYLY--  24-39 16  21 Kabat RSSKNLLHSNGITYLY--  24-39 16  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 186 AbM WYLQKPGQSPQLLIS  40-54 15 186 Kabat WYLQKPGQSPQLLIS  40-54 15 186 Contact --LQKPGQSPQ----  42-50  9 187 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61  7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 188 AbM -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 188 Kabat -GVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 188 Contact SGVPNRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 33 189 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 190 AbM -FGGGTKVEIK 103-112 10 190 Kabat -FGGGTKVEIK 103-112 10 190 Contact TFGGGTKVEIK 102-112 11 191 ¹⁴The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 293) of NMC-H103.VK2.

TABLE 19 mAb NMC-H103.VH3 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat,and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁵ Length NO: HFR1 Chothia EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 192 AbM EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 192 Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT   1-30 30 193 Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF-   1-29 29 194 CDR-H1 Chothia GFTFTHY---  26-32  7  18 AbM GFTFTHYYMS  26-35 10  42 Kabat -----HYYMS  31-35  5  43 Contact ----THYYMS  30-35  6  44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 195 AbM ---WVRQAPGKGLEWLG--  36-49 14 196 Kabat ---WVRQAPGKGLEWLG--  36-49 14 196 Contact ---WVRQAPGKGLE-----  36-46 11 197 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8  19 AbM ---FIRNKAKGYTAE-------  50-61 12  45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19  46 Contact WLGFIRNKAKGYTAE-------  47-61 15  47 HFR3 Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  60-100 41 198 AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  62-100 39 199 Kabat ---------RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR  69-100 32 200 Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTATYYC--  62-98 37 201 CDR-H3 Chothia --DIGDN 101-105  5  20 AbM --DIGDN 101-105  5  20 Kabat --DIGDN 101-105  5  20 Contact ARDIGD-  99-104  6  48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 202 AbM -WGQGTLVTVSS 106-116 11 202 Kabat -WGQGTLVTVSS 106-116 11 202 Contact NWGQGTLVTVSS 105-116 12 203 ¹⁵The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 295) of NMC-H103.VH3.

TABLE 20 mAb NMC-H103.VK3 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat,and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁶ Length NO: LFR1 Chothia DIVMTQTPLSSPVTLGQPASISC------   1-23 23 204 AbM DIVMTQTPLSSPVTLGQPASISC------   1-23 23 204 Kabat DIVMTQTPLSSPVTLGQPASISC------   1-23 23 204 Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL   1-29 29 205 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16  21 AbM RSSKNLLHSNGITYLY--  24-39 16  21 Kabat RSSKNLLHSNGITYLY--  24-39 16  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 206 AbM WYLQKPGQSPQLLIS  40-54 15 206 Kabat WYLQKPGQSPQLLIS  40-54 15 206 Contact --LQKPGQSPQ----  42-50  9 207 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61  7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 208 AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 208 Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 208 Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 33 209 CDR-L3 Chothia AQLLELPYT 94-102  9  23 AbM AQLLELPYT 94-102  9  23 Kabat AQLLELPYT 94-102  9  23 Contact AQLLELPY- 94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 210 AbM -FGGGTKVEIK 103-112 10 210 Kabat -FGGGTKVEIK 103-112 10 210 Contact TFGGGTKVEIK 102-112 11 211 ¹⁶The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 297) of NMC-H103.VK3.

TABLE 21 mAb NMC-H103.VH4 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁷ Length NO: HFR1 Chothia EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 212 AbM EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 212 Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT   1-30 30 213 Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF-   1-29 29 214 CDR-H1 Chothia GFTFTHY---  26-32  7 18 AbM GFTFTHYYMS  26-35 10 42 Kabat -----HYYMS  31-35  5 43 Contact ----THYYMS  30-35  6 44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 215 AbM ---WVRQAPGKGLEWLG--  36-49 14 216 Kabat ---WVRQAPGKGLEWLG--  36-49 14 216 Contact ---WVRQAPGKGLE-----  36-46 11 217 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8 19 AbM ---FIRNKAKGYTAE-------  50-61 12 45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19 46 Contact WLGFIRNKAKGYTAE-------  47-61 15 47 HFR3 Chothia AEYSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR  60-100 41 218 AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR  62-100 39 219 Kabat ---------RFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR  69-100 32 220 Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYC--  62-98 37 221 CDR-H3 Chothia --DIGDN 101-105  5 20 AbM --DIGDN 101-105  5 20 Kabat --DIGDN 101-105  5 20 Contact ARDIGD-  99-104  6 48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 222 AbM -WGQGTLVTVSS 106-116 11 222 Kabat -WGQGTLVTVSS 106-116 11 222 Contact NWGQGTLVTVSS 105-116 12 223 ¹⁷The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 299) of NMC-H103.VH4.

TABLE 22 mAb NMC-H103.VK4 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁸ Length NO: LFR1 Chothia DIVMTQTPLSSPVTLGQPASISC------   1-23 23 224 AbM DIVMTQTPLSSPVTLGQPASISC------   1-23 23 224 Kabat DIVMTQTPLSSPVTLGQPASISC------   1-23 23 224 Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL   1-29 29 225 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16  21 AbM RSSKNLLHSNGITYLY--  24-39 16  21 Kabat RSSKNLLHSNGITYLY--  24-39 16  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 226 AbM WYLQKPGQSPQLLIS  40-54 15 226 Kabat WYLQKPGQSPQLLIS  40-54 15 226 Contact --LQKPGQSPQ----  42-50  9 227 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61  7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 228 AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 228 Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 228 Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 33 229 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 230 AbM -FGGGTKVEIK 103-112 10 230 Kabat -FGGGTKVEIK 103-112 10 230 Contact TFGGGTKVEIK 102-112 11 231 ¹⁸The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 301) of NMC-H103.VK4.

TABLE 23 mAb NMC-H103.VK5 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues¹⁹ Length NO: LFR1 Chothia DIVMTQTPLSSPVTLGQPASISC------   1-23 23 232 AbM DIVMTQTPLSSPVTLGQPASISC------   1-23 23 232 Kabat DIVMTQTPLSSPVTLGQPASISC------   1-23 23 232 Contact DIVMTQTPLSSPVTLGQPASISCRSSKNL   1-29 29 233 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 16  21 AbM RSSKNLLHSNGITYLY--  24-39 16  21 Kabat RSSKNLLHSNGITYLY--  24-39 16  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 234 AbM WYLQKPGQSPQLLIS  40-54 15 234 Kabat WYLQKPGQSPQLLIS  40-54 15 234 Contact --LQKPGQSPQ----  42-50  9 235 CDR-L2 Chothia ----RVSNRAS  55-61  7 236 AbM ----RVSNRAS  55-61  7 236 Kabat ----RVSNRAS  55-61  7 236 Contact LLISRVSNRAS  51-60 10 237 LFR3 Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 238 AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 238 Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 32 238 Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYYC  62-93 33 239 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 240 AbM -FGGGTKVEIK 103-112 10 240 Kabat -FGGGTKVEIK 103-112 10 240 Contact TFGGGTKVEIK 102-112 11 241 ¹⁹The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 303) of NMC-H103.VK5.

TABLE 24 mAb NMC-H103.VH6 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues²⁰ Length NO: HFR1 Chothia EVQLVESGGGLVQPGGSLRLSCTTS-----   1-25 25 242 AbM EVQLVESGGGLVQPGGSLRLSCTTS-----   1-25 25 242 Kabat EVQLVESGGGLVQPGGSLRLSCTTSGFTFT   1-30 30 243 Contact EVQLVESGGGLVQPGGSLRLSCTTSGFTF-   1-29 29 244 CDR-H1 Chothia GFTFTHY---  26-32  7  18 AbM GFTFTHYYMS  26-35 10  42 Kabat -----HYYMS  31-35  5  43 Contact ----THYYMS  30-35  6  44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 245 AbM ---WVRQAPGKGLEWLG--  36-49 14 246 Kabat ---WVRQAPGKGLEWLG--  36-49 14 246 Contact ---WVRQAPGKGLE-----  36-46 11 247 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8  19 AbM ---FIRNKAKGYTAE-------  50-61 12  45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19  46 Contact WLGFIRNKAKGYTAE-------  47-61 15  47 HFR3 Chothia AEYSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR  60-100 41 248 AbM --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR  62-100 39 249 Kabat ---------RFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR  69-100 32 250 Contact --YSASVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYC--  62-98 37 251 CDR-H3 Chothia --DIGDN 101-105  5  20 AbM --DIGDN 101-105  5  20 Kabat --DIGDN 101-105  5  20 Contact ARDIGD-  99-104  6  48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 252 AbM -WGQGTLVTVSS 106-116 11 252 Kabat -WGQGTLVTVSS 106-116 11 252 Contact NWGQGTLVTVSS 105-116 12 253 ²⁰The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 305) of NMC-H103.VH6.

TABLE 2 mAb NMC-H103.VK6 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues²¹ Length NO: LFR1 Chothia DIVMTQTPLSLPVTLGQPASISC------   1-23 23 254 AbM DIVMTQTPLSLPVTLGQPASISC------   1-23 23 254 Kabat DIVMTQTPLSLPVTLGQPASISC------   1-23 23 254 Contact DIVMTQTPLSLPVTLGQPASISCRSSKNL   1-29 29 255 CDR-L1 Chothia RSSKNLLHSNGITYLY----  24-39 10  21 AbM RSSKNLLHSNGITYLY----  24-39 10  21 Kabat RSSKNLLHSNGITYLY----  24-39 10  21 Contact ------LHSNGITYLYWY--  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 256 AbM WYLQKPGQSPQLLIS  40-54 15 256 Kabat WYLQKPGQSPQLLIS  40-54 15 256 Contact --LQKPGQSPQ----  42-50  9 257 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61 7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 258 AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 258 Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 258 Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 33 259 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia FGGGTKVEIK- 103-112 10 260 AbM FGGGTKVEIK- 103-112 10 260 Kabat FGGGTKVEIK- 103-112 10 260 Contact TFGGGTKVEIK 102-112 11 261 ²¹The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 307) of NMC-H103.VK6.

TABLE 26 mAb NMC-H103.VH7 Heavy Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues²² Length NO: HFR1 Chothia EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 262 AbM EVQLVESGGGLVQPGPSLRLSCTTS-----   1-25 25 262 Kabat EVQLVESGGGLVQPGPSLRLSCTTSGFTFT   1-30 30 263 Contact EVQLVESGGGLVQPGPSLRLSCTTSGFTF-   1-29 29 264 CDR-H1 Chothia GFTFTHY---  26-32  7  18 AbM GFTFTHYYMS  26-35 10  42 Kabat -----HYYMS  31-35  5  43 Contact ----THYYMS  30-35  6  44 HFR2 Chothia YMSWVRQAPGKGLEWLGFI  33-51 19 265 AbM ---WVRQAPGKGLEWLG--  36-49 14 266 Kabat ---WVRQAPGKGLEWLG--  36-49 14 266 Contact ---WVRQAPGKGLE-----  36-46 11 267 CDR-H2 Chothia -----RNKAKGYT---------  52-59  8  19 AbM ---FIRNKAKGYTAE-------  50-61 12  45 Kabat ---FIRNKAKGYTAEYSASVKG  50-68 19  46 Contact WLGFIRNKAKGYTAE-------  47-61 15  47 HFR3 Chothia AEYSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR  60-100 41 268 AbM --YSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYCAR  62-100 39 269 Kabat ---------RFTISRDNSQSILYLQMNSLRAEDTAVYYCAR  69-100 32 270 Contact --YSASVKGRFTISRDNSQSILYLQMNSLRAEDTAVYYC--  62-98 37 271 CDR-H3 Chothia --DIGDN 101-105  5  20 AbM --DIGDN 101-105  5  20 Kabat --DIGDN 101-105  5  20 Contact ARDIGD-  99-104  6  48 HFR4 Chothia -WGQGTLVTVSS 106-116 11 272 AbM -WGQGTLVTVSS 106-116 11 272 Kabat -WGQGTLVTVSS 106-116 11 272 Contact NWGQGTLVTVSS 105-116 12 273 ²²The listed residues are residue numbers of the heavy chain variable region (SEQ ID NO: 309) of NMC-H103.VH7.

TABLE 27 mAb NMC-H103.VK7 Light Chain (hu IgG1) CDR and HFR Sequences using Chothia, AbM, Kabat, and Contact CDR definitions: SEQ ID Region Definition Sequence Fragment Residues²³ Length NO: LFR1 Chothia DIVMTQTPLSSPVTLGQSASISC------   1-23 23 274 AbM DIVMTQTPLSSPVTLGQSASISC------   1-23 23 274 Kabat DIVMTQTPLSSPVTLGQSASISC------   1-23 23 274 Contact DIVMTQTPLSSPVTLGQSASISCRSSKNL   1-29 29 275 CDR-L1 Chothia RSSKNLLHSNGITYLY--  24-39 10  21 AbM RSSKNLLHSNGITYLY--  24-39 10  21 Kabat RSSKNLLHSNGITYLY--  24-39 10  21 Contact ------LHSNGITYLYWY  30-41 12  49 LFR2 Chothia WYLQKPGQSPQLLIS  40-54 15 276 AbM WYLQKPGQSPQLLIS  40-54 15 276 Kabat WYLQKPGQSPQLLIS  40-54 15 276 Contact --LQKPGQSPQ----  42-50  9 277 CDR-L2 Chothia ----RVSNLAS  55-61  7  22 AbM ----RVSNLAS  55-61  7  22 Kabat ----RVSNLAS  55-61  7  22 Contact LLISRVSNLA-  51-60 10  50 LFR3 Chothia -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 278 AbM -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 278 Kabat -GVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 32 278 Contact SGVPDRFSGSESGTDFTLKISRVEAEDVGVYFC  62-93 33 279 CDR-L3 Chothia AQLLELPYT  94-102  9  23 AbM AQLLELPYT  94-102  9  23 Kabat AQLLELPYT  94-102  9  23 Contact AQLLELPY-  94-102  8  51 LFR4 Chothia -FGGGTKVEIK 103-112 10 280 AbM -FGGGTKVEIK 103-112 10 280 Kabat -FGGGTKVEIK 103-112 10 280 Contact TFGGGTKVEIK 102-112 11 281 ²³The listed residues are residue numbers of the light chain variable region (SEQ ID NO: 311) of NMC-H103.VK7.

mAb NMC-103 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 132): gaggtgcagctgcaggagtctggaggaggcttggtacagcctgggggttctctgagactctcctg tacaacttctgggttcaccttcactcattactacatgagctgggtccgccagcctccaggcaagg cacttgagtggttgggctttattagaaataaagctaagggttacacagcagagtacagtgcatct gtgaagggtcggttcaccatctccagagataattcccaaagcatcctctatcttcaaatgaacac cctgagacctgaggacagtgccacttattactgtgcaagagatattggggacaactggggtcaag gaaccttagtcaccgtctcctcag mAb NMC-103 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 36) (Complementarity determining regions (CDRs) determined according to the IMGT numbering system are underlined): EVQLQESGGGLVQPGGSLRLSCTTSG FTFTHYY MSWVRQPPGKALEWLGF IRNKAKGYTA EYSAS VKGRFTISRDNSQSILYLQMNTLRPEDSATYYC ARDIGDN WGQGTLVTVSS mAb NMC-103 Light Chain Variable Region DNA Sequence (SEQ ID NO: 134): gatattgtgatgacgcaggctgccttctccaatccagtcactcttggaacatcagcttccatctc ctgcaggtctagtaagaatctcctacatagtaatggcatcacttatttgtattggtatctgcaga ggccaggccagtctcctcagctcctgatatctcgggtgtccaatctggcctcaggagtcccaaac aggttcagtggcagtgagtcaggaactgatttcacactgagaatcagcagagtggaggctgagga tgtgggtgtttatttctgtgctcaactgctagaactcccgtacacgttcggaggggggaccaagt tggaaataaaac mAb NMC-103 Light Chain Variable Region Protein Sequence (SEQ ID NO: 37) (Complementarity determining regions (CDRs) determined according to the IMGT numbering system are underlined): DIVMTQAAFSNPVTLGTSASISCRSS KNLLHSNGITY LYWYLQRPGQSPQLLIS RVS NLASGVPN RFSGSESGTDFTLRISRVEAEDVGVYFC AQLLELPYT FGGGTKLEIK mAb NMC-204 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 136): gaggtgcagctgcaggagtctgggtctgtgctggtgaggcctggagcttcagtgaagctgtcctg caaggcttctggcgacaccctcagcggctcctggatgcactgggcgatgcagaggcctggacaag gccttgagtggattggagagattcatcttaatagaggtactactaactacaatgagaagttcaag ggcaaggccacagtgactgtggacacatcctccagcacagcctacgtggatctcagcagcctgac atctgaggactctgcggtctattactgtgcaagaagcccggggtttgcttactggggccaaggga ctctggtcactgtctctgcag mAb NMC-204 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 38) (Complementarity determining regions (CDRs) determined according to the IMGT numbering system are underlined): EVQLQESGSVLVRPGASVKLSCKAS GDTLSGSW MHWAMQRPGQGLEWIGE IHLNRGTT NYNEKFK GKATVTVDTSSSTAYVDLSSLTSEDSAVYYC ARSPGFA YWGQGTLVTVSA mAb NMC-204 Light Chain Variable Region DNA Sequence (SEQ ID NO: 138): ggcattgtgatgacccaggctgcaccctctgtacctgtcactcctggagagtcagtatccatctc ctgcaggtctagtaagagtctcctgcatagtaatggcaacagttacttgtattggttcctgcaga ggccaggccagtctcctcagctcctgatatatcggatgtccaaccttgcctcaggagtcccagac aggttcagtggcagtgggtcaggaactgctttcacactgagaatcactagagtggaggctgagga tgtgggtgtttattactgtatgcaacatctagaatatcctttcacgttcggctcggggacaaagt tggaaataaaac mAb NMC-204 Light Chain Variable Region Protein Sequence (SEQ ID NO: 39) (Complementarity determining regions (CDRs) determined according to the IMGT numbering system are underlined): GIVMTQAAPSVPVTPGESVSISCRSS KSLLHSNGNSY LYWFLQRPGQSPQLLIY RMS NLASGVPD RFSGSGSGTAFTLRITRVEAEDVGVYYC MQHLEYPFT FGSGTKLEIK mAb NMC-303 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 140): caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctg caaggcttctggctacaccttcaccagctactatatgtactgggtgaagcagaggcctggacaag gccttgagtggattggggggattaatcctaggaatggtggtactaacttcaatgagaagttcaag aacaaggccacactgactgcagacaaatcctccaccacagcctacatgcaactcagtagcctgac atctgaggactctgcggtctattactgtacaagatctggttactatgctatggactattggggtc aaggaacctcagtcaccgtctcctca mAb NMC-303 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 40) (Complementarity determining regions (CDRs) determined according to the Kabat numbering system are underlined): QVQLQQPGAELVKPGASVKLSCKASGYTFT SYYMY WVKQRPGQGLEWIG GINPRNGGTNFNEKFK N KATLTADKSSITAYMQLSSLISEDSAVYYCTR SGYYAMDY WGQGTSVTVSS mAb NMC-303 Light Chain Variable Region DNA Sequence (SEQ ID NO: 142): gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcag ttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactg ttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagt gggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttactt ttgccaacagggtaatacgcttcctcggacgttcggtggaggcaccaagctggaaatcaaa mAb NMC-303 Light Chain Variable Region Protein Sequence (SEQ ID NO: 41) (Complementarity determining regions (CDRs) determined according to the Kabat numbering system are underlined): DIQMTQTTSSLSASLGDRVTISC RASQDISNFLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGS GSGTDYSLTISNLEQEDIATYFC QQGNTLPRT FGGGTKLEIK mAb NMC-C303 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 140): caggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagttgtcctg caaggcttctggctacaccttcaccagctactatatgtactgggtgaagcagaggcctggacaag gccttgagtggattggggggattaatcctaggaatggtggtactaacttcaatgagaagttcaag aacaaggccacactgactgcagacaaatcctccaccacagcctacatgcaactcagtagcctgac atctgaggactctgcggtctattactgtacaagatctggttactatgctatggactattggggtc aaggaacctcagtcaccgtctcctca mAb NMC-C303 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 40) (CDRs determined according to the Kabat numbering system are underlined): QVQLQQPGAELVKPGASVKLSCKASGYTFT SYYMY WVKQRPGQGLEWIG GINPRNGGTNFNEKFK N KATLTADKSSITAYMQLSSLISEDSAVYYCTR SGYYAMDY WGQGTSVTVSS mAb NMC-C303 Light Chain Variable Region DNA Sequence (SEQ ID NO: 142): gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcag ttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactg ttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagt gggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttactt ttgccaacagggtaatacgcttcctcggacgttcggtggaggcaccaagctggaaatcaaa mAb NMC-C303 Light Chain Variable Region Protein Sequence (SEQ ID NO: 41) (CDRs determined according to the Kabat numbering system are underlined): DIQMTQTTSSLSASLGDRVTISC RASQDISNFLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGS GSGTDYSLTISNLEQEDIATYFC QQGNTLPRT FGGGTKLEIK mAb NMC-C103.VH0 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 282): GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGCCTCCAGGCAAGG CACTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACAC CCTGAGGCCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGTCAAG GAACCTTAGTCACCGTCTCCTCA mAb NMC-C103.VH0 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 283) (CDRs determined according to the Kabat numbering system are underlined): EVKLVESGGGLVQPGGSLRLSCTTSGFTFT HYYMS WVRQPPGKALEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSQSILYLQMNTLRPEDSATYYCAR DIGDN WGQGTLVTVSS mAb NMC-C103.VK0 Light Chain Variable Region DNA Sequence (SEQ ID NO: 284): GATATTGTGATGACGCAGGCTGCCTTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA GGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAAAC AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGC TGGAAATAAAA mAb NMC-C103.VK0 Light Chain Variable Region Protein Sequence (SEQ ID NO: 285) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQAAFSNPVTLGTSASISC RSSKNLLHSNGITYLY WYLQRPGQSPQLLIS RVSNLAS GVPN RFSGSESGTDFTLRISRVEAEDVGVYFC AQLLELPYT FGGGTKLEIK mAb NMC-H103.VH1 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 286): GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTTCAAATGAACAC CCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH1 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 287) (CDRs determined according to the Kabat numbering system are underlined): EVKLVESGGGLVQPGPSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSKSTLYLQMNTLRAEDSATYYCAR DIGDN WGQGTLVTVSS mAb NMC-H103.VK1 Light Chain Variable Region DNA Sequence (SEQ ID NO: 288): GATATTGTGATGACGCAGGCTGCCTTCTCCAATCCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGICTCCTCAGCTCCTGATATCTCGGGIGTCCAATCTGGCCTCAGGAGTCCCAAAT AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK1 Light Chain Variable Region Protein Sequence (SEQ ID NO: 289) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQAAFSNPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPN RFSGSESGTDFTLKISRVEAEDVGVYFC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VH2 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 290): GAGGTCAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTTCAAATGAACAG CCTGAGAGCTGAGGACACGGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH2 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 291) (CDRs determined according to the Kabat numbering system are underlined): EVKLVESGGGLVQPGPSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR DIGDN WGQGTLVTVSS mAb NMC-H103.VK2 Light Chain Variable Region DNA Sequence (SEQ ID NO: 292): GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAAAT AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK2 Light Chain Variable Region Protein Sequence (SEQ ID NO: 293) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQTPLSSPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPN RFSGSESGTDFTLKISRVEAEDVGVYFC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VE3 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 294): GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTTCAAATGAACAG CCTGAGAGCTGAGGACACGGCCACTTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH3 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 295) (CDRs determined according to the Kabat numbering system are underlined): EVQLVESGGGLVQPGPSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSKSTLYLQMNSLRAEDTATYYCAR DIGDN WGQGTLVTVSS mAb NMC-H103.VK3 Light Chain Variable Region DNA Sequence (SEQ ID NO: 296): GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC AGGTICAGTGGCTCCGAGTCAGGAACTGATTICACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK3 Light Chain Variable Region Protein Sequence (SEQ ID NO: 297) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQTPLSSPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPD RFSGSESGTDFTLKISRVEAEDVGVYFC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VH4 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 298): GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTTCAAATGAACAG CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH4 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 299) (CDRs) determined according to the Kabat numbering system are underlined): EVQLVESGGGLVQPGPSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSKSTLYLQMNSLRAEDTAVYYCARDIGDNWGQGTLVTVSS mAb NMC-H103.VK4 Light Chain Variable Region DNA Sequence (SEQ ID NO: 300): GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTACTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK4 Light Chain Variable Region Protein Sequence (SEQ ID NO: 301) (CDRs) determined according to the Kabat numbering system are underlined): DIVMTQTPLSSPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPD RFSGSESGTDFTLKISRVEAEDVGVYYC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VK5 Light Chain Variable Region DNA Sequence (SEQ ID NO: 302): GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCGGGCCTCAGGAGTCCCAGAC AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTACTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK5 Light Chain Variable Region Protein Sequence (SEQ ID NO: 303) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQTPLSSPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNRAS GVPD RFSGSESGTDFTLKISRVEAEDVGVYYC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VH6 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 304): GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCAAGAGCACGCTCTATCTTCAAATGAACAG CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH6 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 305) (CDRs determined according to the Kabat numbering system are underlined): EVQLVESGGGLVQPGGSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSKSTLYLQMNSLRAEDTAVYYCAR DIGDN WGQGTLVTVSS mAb NMC-H103.VK6 Light Chain Variable Region DNA Sequence (SEQ ID NO: 306): GATATTGTGATGACGCAGACTCCACTCTCCCTGCCAGTCACTCTTGGACAGCCGGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK6 Light Chain Variable Region Protein Sequence (SEQ ID NO: 307) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQTPLSLPVTLGQPASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPD RFSGSESGTDFTLKISRVEAEDVGVYFC AQLLELPYT FGGGTKVEIK mAb NMC-H103.VH7 Heavy Chain Variable Region DNA Sequence (SEQ ID NO: 308): GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGCCGTCTCTGAGACTCTCCTG TACAACTTCTGGGTTCACCTTCACTCATTACTACATGAGCTGGGTCCGCCAGGCTCCAGGCAAGG GGCTTGAGTGGTTGGGCTTTATTAGAAATAAAGCTAAGGGTTACACAGCAGAGTACAGTGCATCT GTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACAG CCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCAAGAGATATTGGGGACAACTGGGGCCAAG GAACCCTGGTCACCGTCTCCTCA mAb NMC-H103.VH7 Heavy Chain Variable Region Protein Sequence (SEQ ID NO: 309) (CDRs determined according to the Kabat numbering system are underlined): EVQLVESGGGLVQPGPSLRLSCITSGFTFT HYYMS WVRQAPGKGLEWLG FIRNKAKGYTAEYSAS VKG RFTISRDNSQSILYLQMNSLRAEDTAVYYCAR DIGDN WGQGTLVTVSS mAb NMC-H103.VK7 Light Chain Variable Region DNA Sequence (SEQ ID NO: 310): GATATTGTGATGACGCAGACTCCACTCTCCTCACCAGTCACTCTTGGACAGTCAGCTTCCATCTC CTGCAGGTCTTCCAAGAATCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGA AGCCAGGCCAGTCTCCTCAGCTCCTGATATCTCGGGTGTCCAATCTGGCCTCAGGAGTCCCAGAC AGGTTCAGTGGCTCCGAGTCAGGAACTGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTGTTTATTTCTGTGCTCAACTGCTAGAACTCCCGTACACGTTCGGAGGGGGGACCAAGG TGGAAATCAAA mAb NMC-H103.VK7 Light Chain Variable Region Protein Sequence (SEQ ID NO: 311) (CDRs determined according to the Kabat numbering system are underlined): DIVMTQTPLSSPVTLGQSASISC RSSKNLLHSNGITYLY WYLQKPGQSPQLLIS RVSNLAS GVPD RFSGSESGTDFTLKISRVEAEDVGVYFC AQLLELPYT FGGGTKVEIK mAb NMC-C303 Heavy Chain Protein Sequence (SEQ ID NO: 312): MGWSCIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLE WIGGINPRNGGINFNEKFKNKATLTADKSSTTAYMQLSSLTSEDSAVYYCTRSGYYAMDYWGQGT SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK mAb NMC-C303 Light Chain Protein Sequence (SEQ ID NO: 313): MGWSCIILFLVATATGVHSDIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKL LIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

INCORPORATION BY REFERENCE

Various references such as patents, patent applications, and publications are cited herein, the disclosures of which are hereby incorporated by reference herein in their entireties. 

What is claimed is:
 1. A humanized antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, or a VH having at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and/or (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311, or a VL having at least 95% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:303; SEQ ID NO:307, and SEQ ID NO:311.
 2. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:287, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:287.
 3. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:291, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:291.
 4. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:295, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:295.
 5. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:299, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:299.
 6. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:305, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:305.
 7. The humanized antibody or fragment of claim 1, which comprises a VH having the amino acid sequence of SEQ ID NO:309, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:309.
 8. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:289, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:289.
 9. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:293, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:293.
 10. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:297, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:297.
 11. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:301, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:301.
 12. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:303, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:303.
 13. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:307, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:307.
 14. The humanized antibody or fragment of any one of claims 1-7, which comprises a VL having the amino acid sequence of SEQ ID NO:311, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:311.
 15. The humanized antibody or fragment of claim 1, which comprises: (i) a VH having the amino acid sequence of SEQ ID NO:299, and (ii) a VL having the amino acid sequence of SEQ ID NO:297.
 16. The humanized antibody or fragment of claim 1, which comprises: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, and SEQ ID NO:299, and (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, and SEQ ID NO:303.
 17. The humanized antibody or fragment of claim 1, which comprises: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (ii) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:297, SEQ ID NO:307, and SEQ ID NO:311.
 18. An antibody or a fragment comprising a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); or (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51).
 19. The antibody or fragment of claim 18, which comprises a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).
 20. An antibody or a fragment thereof that specifically binds to HDM2 or MDM2, said antibody or fragment comprising: (a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO:283, or a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:283, and (ii) a light chain variable region (VL) having an amino acid sequence of SEQ ID NO:285, or a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:285.
 21. A chimeric antibody that specifically binds to HDM2 or MDM2, said antibody comprising: (a) a heavy chain having an amino acid sequence of SEQ ID NO:312, or a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:312, and/or (b) a light chain having an amino acid sequence of SEQ ID NO:313, or a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:313.
 22. The antibody or fragment of any one of claims 1-21, wherein the antibody is a monoclonal antibody.
 23. The antibody or fragment of any one of claims 1-20, which is an immunoglobulin.
 24. The antibody or fragment of claim 23, wherein the immunoglobulin is an IgG.
 25. The antibody or fragment of claim 24, wherein the immunoglobulin is of IgG1 isotype.
 26. The antibody or fragment of any one of claims 1-20, which comprises an Fc region, and wherein the Fc region is a human IgG1, a human IgG2, a human IgG3, a human IgG4, or a human IgM Fc region.
 27. The antibody or fragment of claim 26, wherein the Fc region is a human IgG1.
 28. The antibody or fragment of any one of claims 1-27, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytoxicity (ADCC).
 29. The antibody or fragment of claim 28, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC).
 30. The antibody or fragment of any one of claims 1-20, which is an Fv fragment, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a single chain antibody molecule, or a single chain Fv (scFv).
 31. The antibody or fragment of any one of claims 1-30, wherein the HDM2 is an HDM2 variant that lacks a nuclear localization signal domain, an HDM2 variant that lacks the sequence of amino acids 179 to 185 of SEQ ID NO:4, and/or an HDM2 variant that lacks the sequence of amino acids 464 to 471 of SEQ ID NO:4.
 32. The antibody or fragment of any one of claims 1-31, which is purified.
 33. The antibody or fragment of any one of claims 1-32, which inhibits tumor cell proliferation in vivo.
 34. An antibody-drug conjugate comprising the antibody or fragment of any one of claims 1-33, bound to a cytotoxic drug.
 35. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or fragment of any one of claims 1-33 or the antibody-drug conjugate of claim
 34. 36. A method of treating cancer in a subject in need thereof, said method comprising administering to the subject: (i) the antibody or fragment of any one of claims 1-33, (ii) the antibody-drug conjugate of claim 34, or (iii) the pharmaceutical composition of claim
 35. 37. A method of inhibiting or preventing metastasis in a subject having a cancer, said method comprising administering to the subject: (i) the antibody or fragment of any one of claims 1-33, (ii) the antibody-drug conjugate of claim 34, or (iii) the pharmaceutical composition of claim
 35. 38. A method of preventing cancer recurrence or preventing cancer relapse in a subject in need thereof, said method comprising administering to the subject: (i) the antibody or fragment of any one of claims 1-33, (ii) the antibody-drug conjugate of claim 34, or (iii) the pharmaceutical composition of claim
 35. 39. A method of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, said method comprising administering to the subject: (i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) the antibody or fragment of any one of claims 1-33, (iv) the antibody-drug conjugate of claim 34, or (v) the pharmaceutical composition of claim
 35. 40. The method of claim 39, wherein the one or more inhibitory checkpoint molecules are selected from the group consisting of: CTLA-4, PD-1, PD-L1, and PD-L2.
 41. The method of claim 39, wherein the one or more inhibitory checkpoint molecules is PD-1.
 42. The method of claim 39, wherein the inhibitor of one or more inhibitory checkpoint molecules is an inhibitory antibody to PD-1.
 43. The method of any one of claims 36-42, wherein said method comprises administering to the subject the antibody or fragment of any one of claims 1-33.
 44. The method of any one of claims 36-42, said method comprising administering to the subject the pharmaceutical composition of claim
 35. 45. The method of claim 43 or 44, wherein the antibody or fragment is not bound to a cell-penetrating peptide.
 46. The method of any one of claims 36-42, said method comprising administering to the subject the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
 47. The method of any one of claims 36-42, said method comprising administering to the subject the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component.
 48. The method of claim 46 or 47, wherein the antibody or fragment is a humanized antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
 49. The method of claim 46 or 47, wherein the antibody or fragment is a human antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
 50. The method of claim 46 or 47, wherein the antibody or fragment is a chimeric antibody.
 51. The method of any one of claims 46-50, wherein the antibody or fragment is a monoclonal antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4.
 52. The method of any one of claims 46-51, wherein the antibody or fragment is an immunoglobulin.
 53. The method of claim 52, wherein the immunoglobulin is an IgG.
 54. The method of claim 53, wherein the immunoglobulin is of IgG1 isotype.
 55. The method of claim 53, wherein the immunoglobulin is of IgG3 isotype.
 56. The method of any one of claims 46-51, wherein the antibody or fragment comprises an Fc region, and wherein the Fc region is a human IgG1, a human IgG2, a human IgG3, a human IgG4, or a human IgM Fc region.
 57. The method of any one of claims 46-56, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytoxicity (ADCC).
 58. The method of claim 57, wherein the antibody or fragment mediates complement-dependent cytotoxicity (CDC).
 59. The method of any one of claims 46-51, wherein the antibody or fragment is an Fv fragment, a Fab fragment, a Fab′ fragment, a F(ab′) 2 fragment, a single chain antibody molecule, or a single chain Fv (scFv).
 60. The method of any one of claims 46-59, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1).
 61. The method of any one of claims 46-59, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is TTSQIPASEQE (SEQ ID NO:2).
 62. The method of any one of claims 46-59, wherein the antibody or fragment specifically binds to a peptide, wherein the sequence of the peptide is CPVCRQPIQMIVLTYFP (SEQ ID NO:3).
 63. The method of any one of claims 46-59, wherein the antibody or a fragment comprises a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GFTFTHY (SEQ ID NO:18), the VH CDR 2 has the amino acid sequence RNKAKGYT (SEQ ID NO:19), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (ii) the VH CDR 1 has the amino acid sequence GFTFTHYYMS (SEQ ID NO:42), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAE (SEQ ID NO:45), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iii) the VH CDR 1 has the amino acid sequence HYYMS (SEQ ID NO:43), the VH CDR 2 has the amino acid sequence FIRNKAKGYTAEYSASVKG (SEQ ID NO:46), and the VH CDR 3 has the amino acid sequence DIGDN (SEQ ID NO:20); (iv) the VH CDR 1 has the amino acid sequence THYYMS (SEQ ID NO:44), the VH CDR 2 has the amino acid sequence WLGFIRNKAKGYTAE (SEQ ID NO:47), and the VH CDR 3 has the amino acid sequence ARDIGD (SEQ ID NO:48); or (v) the VH CDR 1 has the amino acid sequence FTFTHYY (SEQ ID NO:144), the VH CDR 2 has the amino acid sequence IRNKAKGYTA (SEQ ID NO:145), and the VH CDR 3 has the amino acid sequence ARDIGDN (SEQ ID NO:146).
 64. The method of any one of claims 46-59, wherein the antibody or a fragment comprises a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GDTLSGS (SEQ ID NO:24), the VH CDR 2 has the amino acid sequence HLNRGT (SEQ ID NO:25), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (ii) the VH CDR 1 has the amino acid sequence GDTLSGSWMH (SEQ ID NO:52), the VH CDR 2 has the amino acid sequence EIHLNRGTTN (SEQ ID NO:55), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iii) the VH CDR 1 has the amino acid sequence GSWMH (SEQ ID NO:53), the VH CDR 2 has the amino acid sequence EIHLNRGTTNYNEKFKG (SEQ ID NO:56), and the VH CDR 3 has the amino acid sequence SPGFAY (SEQ ID NO:26); (iv) the VH CDR 1 has the amino acid sequence SGSWMH (SEQ ID NO:54), the VH CDR 2 has the amino acid sequence WIGEIHLNRGTTN (SEQ ID NO:57), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58); or (v) the VH CDR 1 has the amino acid sequence GDTLSGSW (SEQ ID NO:148), the VH CDR 2 has the amino acid sequence IHLNRGTT (SEQ ID NO:143), and the VH CDR 3 has the amino acid sequence ARSPGFA (SEQ ID NO:58).
 65. The method of any one of claims 46-59, wherein the antibody or a fragment comprises a heavy chain variable region (VH) comprising VH complementarity determining region (“CDR”) 1, VH CDR 2, and VH CDR 3, wherein: (i) the VH CDR 1 has the amino acid sequence GYTFTSY (SEQ ID NO:30), the VH CDR 2 has the amino acid sequence NPRNGG (SEQ ID NO:31), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (ii) the VH CDR 1 has the amino acid sequence GYTFTSYYMY (SEQ ID NO:62), the VH CDR 2 has the amino acid sequence GINPRNGGTN (SEQ ID NO:65), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); (iii) the VH CDR 1 has the amino acid sequence SYYMY (SEQ ID NO:63), the VH CDR 2 has the amino acid sequence GINPRNGGTNFNEKFKN (SEQ ID NO:66), and the VH CDR 3 has the amino acid sequence SGYYAMDY (SEQ ID NO:32); or (iv) the VH CDR 1 has the amino acid sequence TSYYMY (SEQ ID NO:64), the VH CDR 2 has the amino acid sequence WIGGINPRNGGTN (SEQ ID NO:67), and the VH CDR 3 has the amino acid sequence TRSGYYAMD (SEQ ID NO:68).
 66. The method of claim 63, wherein the antibody or fragment further comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNLAS (SEQ ID NO:22), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNLA (SEQ ID NO:50), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51); or (iii) the VL CDR 1 has the amino acid sequence KNLLHSNGITY (SEQ ID NO:147), the VL CDR 2 has the amino acid sequence RVS, and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23).
 67. The method of claim 63, wherein the antibody or fragment comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKNLLHSNGITYLY (SEQ ID NO:21), the VL CDR 2 has the amino acid sequence RVSNRAS (SEQ ID NO:236), and the VL CDR 3 has the amino acid sequence AQLLELPYT (SEQ ID NO:23); or (ii) the VL CDR 1 has the amino acid sequence LHSNGITYLYWY (SEQ ID NO:49), the VL CDR 2 has the amino acid sequence LLISRVSNRAS (SEQ ID NO:237), and the VL CDR 3 has the amino acid sequence AQLLELPY (SEQ ID NO:51).
 68. The method of claim 64, wherein the antibody or fragment further comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RSSKSLLHSNGNSYLY (SEQ ID NO:27), the VL CDR 2 has the amino acid sequence RMSNLAS (SEQ ID NO:28), and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29); (ii) the VL CDR 1 has the amino acid sequence LHSNGNSYLYWF (SEQ ID NO:59), the VL CDR 2 has the amino acid sequence LLIYRMSNLA (SEQ ID NO:60), and the VL CDR 3 has the amino acid sequence MQHLEYPF (SEQ ID NO:61); or (iii) the VL CDR 1 has the amino acid sequence KSLLHSNGNSY (SEQ ID NO:141), the VL CDR 2 has the amino acid sequence RMS, and the VL CDR 3 has the amino acid sequence MQHLEYPFT (SEQ ID NO:29).
 69. The method of claim 65, wherein the antibody or fragment further comprises a light chain variable region (VL) comprising VL complementarity determining region (“CDR”) 1, VL CDR 2, and VL CDR 3, wherein: (i) the VL CDR 1 has the amino acid sequence RASQDISNFLN (SEQ ID NO:33), the VL CDR 2 has the amino acid sequence YTSRLHS (SEQ ID NO:34), and the VL CDR 3 has the amino acid sequence QQGNTLPRT (SEQ ID NO:35); or (ii) the VL CDR 1 has the amino acid sequence SNFLNWY (SEQ ID NO:69), the VL CDR 2 has the amino acid sequence LLIYYTSRLH (SEQ ID NO:70), and the VL CDR 3 has the amino acid sequence QQGNTLPR (SEQ ID NO:71).
 70. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 19 to 50 of SEQ ID NO:4.
 71. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 154 to 167 of SEQ ID NO:4.
 72. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 1 to 60 of SEQ ID NO:4.
 73. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 26 to 60 of SEQ ID NO:4.
 74. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 101 to 200 of SEQ ID NO:4.
 75. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within amino acids 50 to 60 of SEQ ID NO:4.
 76. The method of any one of claims 46-59, wherein the extracellularly accessible epitope of M(H)DM2/4 is within the terminal 60 amino acids at the C-terminus of the HDM2 on the plasma membrane of the cancer cells.
 77. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody OP145.
 78. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody 965 (SMP14).
 79. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-813 (N-20).
 80. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with rabbit anti-HDM2 antibody sc-812 (C-18).
 81. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with mouse anti-HDM2 antibody M01, clone 1A7.
 82. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds to HDM2 or MDM2, said antibody or fragment comprising: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, and SEQ ID NO:299, and (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, and SEQ ID NO:303.
 83. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with a humanized antibody or a fragment that specifically binds to HDM2 or MDM2, said antibody or fragment comprising: (a) a heavy chain variable region (VH) comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:299, SEQ ID NO:305, and SEQ ID NO:309, and (b) a light chain variable region (VL) comprising a VL having an amino acid sequence selected from the group consisting of SEQ ID SEQ ID NO:297, SEQ ID NO:307, and SEQ ID NO:311.
 84. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with an antibody or a fragment that specifically binds to HDM2 or MDM2, said antibody or fragment comprising: (a) a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO:283, and (ii) a light chain variable region (VL) having an amino acid sequence of SEQ ID NO:285.
 85. The method of any one of claims 46-59, wherein the antibody or fragment competes for binding to M(H)DM2/4 with a chimeric antibody that specifically binds to HDM2 or MDM2, said antibody comprising: (a) a heavy chain having an amino acid sequence of SEQ ID NO:312, and (ii) a light chain having an amino acid sequence of SEQ ID NO:313.
 86. The method of any one of claims 46-85, wherein the M(H)DM2/4 is an HDM2 variant that lacks a nuclear localization signal domain, an HDM2 variant that lacks the sequence of amino acids 179 to 185 of SEQ ID NO:4, and/or an HDM2 variant that lacks the sequence of amino acids 464 to 471 of SEQ ID NO:4.
 87. The method of any one of claims 46-86, wherein the antibody or fragment is purified.
 88. The method of any one of claims 36-42, said method comprising administering to the subject an antibody-drug conjugate, said antibody-drug conjugate comprising the antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide.
 89. The method of any one of claims 36-87, wherein the antibody or fragment is not bound to a cytotoxic drug.
 90. The method of any one of claims 36-89, wherein the cancer is a type of cancer that is known to metastasize.
 91. The method of any one of claims 36-90, wherein the cancer is an advanced stage cancer.
 92. The method of any one of claims 36-91, wherein the cancer is a metastatic cancer.
 93. The method of any one of claims 36-92, wherein the cancer is a solid cancer.
 94. The method of claim 93, wherein the cancer is a lung cancer, a cervical cancer, an endometrial cancer, an ovarian cancer, a pancreatic cancer, a melanoma, a breast cancer, a colon cancer, a bladder cancer, an astrocytic neoplasm, a glioblastoma, or a pediatric Rhabdomyosarcoma.
 95. The method of claim 94, wherein the cancer is a pancreatic cancer, a lung cancer, or a colon cancer.
 96. The method of claim 93, wherein the cancer is a bladder cancer, a breast cancer, a lung cancer, a colon cancer, a melanoma, a sarcoma, an ovarian cancer, a glioma, a head and neck cancer, an urothelial cancer, a pancreatic cancer, a squamous cell carcinoma of the hypopharynx.
 97. The method of claim 96, wherein the breast cancer is a triple negative breast cancer.
 98. The method of claim 96, wherein the sarcoma is an endometrial stromal sarcoma.
 99. The method of any one of claims 36-92, wherein the cancer is a non-solid cancer.
 100. The method of claim 99, wherein the cancer is a leukemia or a lymphoma.
 101. The method of any one of claims 36-100, wherein the antibody or fragment is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or intratumorally.
 102. The method of any one of claims 36-101, further comprising administering to the subject a cancer therapy different from said antibody or fragment or antibody-drug conjugate.
 103. The method of claim 102, wherein the cancer therapy is a chemotherapy.
 104. The method of claim 103, wherein the chemotherapy is gemcitabine.
 105. The method of claim 103, wherein the chemotherapy is nab-paclitaxel.
 106. The method of claim 103, wherein the chemotherapy is cisplatin.
 107. The method of claim 103, wherein the chemotherapy is 5-FU.
 108. The method of claim 103, wherein the chemotherapy is paclitaxel.
 109. The method of claim 103, wherein the cancer is a pancreatic cancer, and wherein the chemotherapy is a combination of gemcitabine and nab-paclitaxel.
 110. The method of claim 109, wherein the gemcitabine and nab-paclitaxel are administered in doses that are lower than doses used when gemcitabine and nab-paclitaxel are administered not in combination with an anti-cancer antibody.
 111. The method of claim 109, wherein the subject is human, and wherein the gemcitabine is administered in a dose that is equal to or less than 1,000 mg/m2, and the nab-paclitaxel is administered in a dose that is equal to or less than 125 mg/m2.
 112. The method of claim 109, wherein the subject is human, and wherein gemcitabine is administered in a dose that is equal to or less than 500 mg/m2, and the nab-paclitaxel is administered in a dose that is equal to or less than 62.5 mg/m2.
 113. The method of any one of claims 109-112, wherein the combination of gemcitabine and nab-paclitaxel is administered with a frequency of every 2 weeks or less.
 115. The method of any one of claims 36-113, wherein the subject is a human.
 116. The method of claim 102, wherein the cancer therapy is an immunotherapy.
 117. The method of claim 116, wherein the immunotherapy is an inhibitor of one or more inhibitory checkpoint molecules.
 118. The method of claim 117, wherein the one or more inhibitory checkpoint molecules are selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, OX40, and LAG-3.
 119. The method of claim 102, wherein the cancer therapy is an inhibitor of one or more of: EGFR, KRAS, STK11, ALK, BRAF, ERBB2, RET, ROS1, B2M, HLA, POLE, IGF-1, ERK/MAPK, PI3K/AKT, TGF-β, DNMT3A, IFNγ, JAK1/JAK2/JAK3, CD274, PTEN, ART, and CDK.
 120. The method of claim 102, wherein the cancer therapy is a peptide inhibitor of p53-M(H)DM2/4 interaction.or a small molecule inhibitor of p53-M(H)DM2/4 interaction.
 121. The method of any one of claims 36-101, wherein the antibody or fragment is administered as a monotherapy.
 122. A method of selecting and treating a subject having a cancer, said method comprising: (a) identifying a subject having a cancer wherein an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of an intact cell of the cancer; and (b) administering to the subject: (i) the antibody or fragment of any one of claims 1-33, (ii) the antibody-drug conjugate of claim 34, or (iii) the pharmaceutical composition of claim
 35. 123. The method of claim 122, which further comprises before step (b) a step of determining whether the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer, wherein the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 is the antibody or fragment of any one of claims 1-33.
 124. The method of claim 123, which further comprises before the determining step the step of obtaining intact cells of the cancer.
 125. A method of diagnosing cancer in a subject, said method comprising: (a) detecting whether the antibody or fragment of any one of claims 1-33 binds to the surface of intact cells of the subject; and (b) diagnosing the subject with cancer if binding is detected in step (a).
 126. The method of claim 125, which is an ex vivo method.
 127. The method of claim 125, further comprising obtaining intact cells from the subject before step (a).
 128. The method of claim 125, which comprises administering the antibody or fragment to the subject before the detecting in step (a), and wherein the detecting is performed by in vivo imaging of the subject.
 129. The method of any one of claims 122-128, wherein, in step (a), the antibody or fragment is labeled.
 130. The method of any one of claims 122-129, wherein the subject is a human.
 131. The antibody or a fragment thereof of any one of claims 1-33, wherein the antibody or fragment specifically binds to an extracellularly accessible epitope of HDM2.
 132. The method of any one of claims 36-130, said method comprising administering to the subject the antibody or fragment that specifically binds to an extracellularly accessible epitope of HDM2.
 133. A method of treating cancer in a subject who has experienced an accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, said method comprising: (a) identifying a subject who (i) has experienced accelerated rate of cancer growth in response to administration to the subject of an inhibitor of one or more inhibitory checkpoint molecules, and (ii) has a cancer wherein an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer; and (b) administering to the subject: (i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) the antibody or fragment of any one of claims 1-33, (iv) the antibody-drug conjugate of claim 34, or (v) the pharmaceutical composition of claim
 35. 134. The method of claim 133, which further comprises before step (b) a step of determining whether the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer, wherein the antibody or fragment is the antibody or fragment of any one of claims 1-33.
 135. The method of claim 134, which further comprises before the determining step the step of obtaining intact cells of the cancer.
 136. The method of any one of claims 133-135, wherein step (a) further comprises selecting a subject who has a gene amplification of M(H)DM2/4.
 137. The method of any one of claims 133-136, wherein step (a) further comprises selecting a subject who has an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells.
 138. The method of any one of claims 133-137, wherein step (a) further comprises selecting a subject who has a cancer wherein there is an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to the binding of the antibody or fragment thereof to the surface of intact normal cells.
 139. A method of treating cancer in a subject who is a hyper-progressor in response to administration of an inhibitor of one or more inhibitory checkpoint molecules, said method comprising: (a) identifying a subject, wherein the subject has (i) a cancer wherein an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer, and (ii) a gene amplification of M(H)DM2/4, or an increased protein expression of M(H)DM2/4 in the cells of the cancer relative to the level of protein expression of M(H)DM2/4 in normal cells; and (b) administering to the subject: (i) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, or an antibody-drug conjugate comprising the antibody or fragment bound to a cytotoxic drug, wherein said antibody or fragment is not bound to a cell-penetrating peptide, (ii) an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4, wherein said antibody or fragment is not bound to a cytotoxic component, (iii) the antibody or fragment of any one of claims 1-33, (iv) the antibody-drug conjugate of claim 34, or (v) the pharmaceutical composition of claim
 35. 140. The method of claim 139, which further comprises before step (b) a step of determining whether the antibody or fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer, wherein the antibody or fragment is the antibody or fragment of any one of claims 1-33.
 141. The method of claim 133, which further comprises before the determining step the step of obtaining intact cells of the cancer.
 142. The method of any one of claims 139-141, wherein step (a) further comprises selecting a subject who has a gene amplification of M(H)DM2/4.
 143. The method of any one of claims 139-142, wherein step (a) further comprises selecting a subject who has an increased binding of an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 to the surface of intact cells of the cancer relative to its binding to the surface of intact normal cells.
 144. A method of diagnosing a hyper-progressive disease in a subject who has cancer, said method comprising: (a) determining whether a gene amplification of M(H)DM2/4 is present in the cells of the cancer of the subject; (b) determining whether an antibody or a fragment thereof that specifically binds to an extracellularly accessible epitope of M(H)DM2/4 binds to the surface of intact cells of the cancer of the subject; and (c) diagnosing the subject with the hyper-progressive disease if the gene amplification is determined to be present in step (a) and binding is detected in step (b).
 145. The method of claim 144, which is an ex vivo method.
 146. The method of claim 144 or 145, further comprising obtaining intact cells from the subject before step (b).
 147. The method of claim 144, which comprises administering the antibody or fragment to the subject before the detecting in step (b), and wherein the detecting is performed by in vivo imaging of the subject.
 148. The method of any one of claims 144-147, wherein, in step (b), the antibody or fragment is labeled.
 149. The method of any one of claims 144-148, wherein the antibody or fragment is the antibody or fragment of any one of claims 1-33.
 150. The method of any one of claims 133-149, wherein the subject is a human.
 151. A vaccine composition comprising: (i) an immunogenic amount of a peptide, wherein the amino acid sequence of the peptide is MCNTNMSVPTDGAVT (SEQ ID NO:1), TTSQIPASEQE (SEQ ID NO:2), or CPVCRQPIQMIVLTYFP (SEQ ID NO:3), or a polynucleotide encoding the peptide; and (ii) a pharmaceutically acceptable carrier.
 152. The vaccine composition of claim 151, wherein the peptide is purified.
 153. The vaccine composition of claim 151 or 152, further comprising an adjuvant.
 154. A method of vaccinating a subject at risk for developing cancer or a subject who has been diagnosed with cancer by administering to the subject the vaccine composition of any one of claims 151-153. 